Mitrecin a polypeptide with antimicrobial activity

ABSTRACT

The present invention provides a Mitrecin A polypeptide useful in prevention and treatment of one or more bacteria. Also provided is a method to kill or prevent growth of one or more bacteria comprising contacting the one or more bacteria with a Mitrecin A polypeptide. The target bacteria can be selected from the group consisting of a Gram-positive bacterium, a Gram-negative bacterium, or both. In one embodiment, the present invention is drawn to a polynucleotide encoding a Mitrecin A polypeptide, a vector comprising the polynucleotide, a host cell comprising the polynucleotide, or a composition comprising the Mitrecin A polypeptide, the polynucleotide, the vector, or the host cell.

CROSS REFERENCE TO RELATED APPLICATIONS

The following applications are incorporated by reference in theirentirety: U.S. patent application Ser. No. 14/828,127, filed Aug. 17,2015, now U.S. Pat. No. 9,562,255; U.S. patent application Ser. No.14/025,360, filed Sep. 12, 2013, now U.S. Pat. No. 9,139,626; and U.S.Provisional Application No. 61/700,514, filed Sep. 13, 2012.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEBSECTION

The content of the electronically submitted sequence listing (Name:2272_1810003_SeqListing_ST25.txt, Size: 7,461 bytes; and Date ofCreation: Jan. 3, 2017) is submitted herewith and is incorporated hereinby reference in its entirety.

BACKGROUND OF THE INVENTION Field of Invention

Bacteriocins are defined as compounds produced by bacteria that have abiologically active protein moiety and bactericidal action (Tagg et al.,Bacteriological Reviews, Volume 40, 722-256, 1976). Theircharacteristics may include: (1) a narrow inhibitory spectrum ofanti-microbial activity among closely related species; (2) attachment tospecific cell receptors; and/or (3) plasmid-borne genetic determinantsof bacteriocin production and of host cell bacteriocin immunity. Theirwidespread occurrence in bacterial species isolated from complexmicrobial communities such as the intestinal tract, the oral or otherepithelial surfaces, suggests that bacteriocins may have a regulatoryrole in terms of population dynamics within bacterial ecosystems.

Bacteriocins have commercial uses in the control of microbes such asextending the shelf life of foods, decreasing spoilage, and reducing therisk of exposure to food-borne pathogens. Additionally, as bacteriocinstypically act on the cell membrane, there is no cross-resistance withapproved and marketed antibiotics. Moreover, bacteriocins targetprokaryotes and in some cases eukaryotes, making them safe for humanconsumption. Bacteriocins can also reduce the use of chemicalpreservatives, as well as facilitate the marketing of foods that areless acidic, have a lower salt content, or have a higher water contentthan foods now available.

To date, only one such bacteriocin, nisin, has been approved for use bythe United States Food and Drug Administration (FDA) as an antimicrobialagent for use on casings for frankfurters and on cooked meat and poultryproducts. However, nisin-resistant L. monocytogenes strains have beenreported. In addition, different bacteriocins have different spectra ofbacteriocidal activity. Therefore, there is a need in the art for asafe, new broad-spectrum bacteriocin.

SUMMARY OF THE INVENTION

The present invention provides an isolated polypeptide comprising anamino acid sequence at least 90% identical to SEQ ID NO: 2 or a fragmentthereof, wherein the polypeptide kills or inhibits growth of one or morebacteria (e.g., Mitrecin A). The one or more bacteria can be aGram-positive bacterium, a Gram-negative bacterium, or both aGram-positive bacterium and a Gram-negative bacterium. The one or morebacteria are, for example, Vibrio cholerae, Shigella sonnei, Aeromonashydrophila, Salmonella enterica, Yersinia pseudotuberculosis, andBacillus subtilis.

In one embodiment, the isolated polypeptide comprises an amino acidsequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ IDNO: 2. In another embodiment, the polypeptide comprises an amino acidsequence is encoded by a nucleotide sequence at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1.

In some embodiment, the isolated polypeptide of the invention comprisesConserved Region 1 of SEQ ID NO: 2, wherein the polypeptide kills orinhibits growth of one or more bacteria. In one aspect, Conserved Region1 comprises amino acids 54 to 73 of SEQ ID NO: 2. In another aspect, thepolypeptide further comprises Conserved Region 2 (e.g., amino acids 81to 90 of SEQ ID NO: 2). In other aspects, the polypeptide comprisesConserved Region 1, Conserved Region 2, and Conserved Region 3 (e.g.,amino acids 106 to 121 of SEQ ID NO: 2). In some aspects, thepolypeptide comprises amino acids 54 to 121 of SEQ ID NO: 2.

In certain embodiments, the polypeptide is conjugated to a heterologousmoiety.

In other embodiments, the invention includes an isolated polynucleotidecomprising a nucleic acid sequence encoding a Mitrecin A polypeptide.The polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, or 100% identical to SEQ ID NO: 1.

In still other embodiments, the invention includes a vector comprising aMitrecin A polynucleotide or a host cell comprising the vector.

In further embodiments, the invention provides a composition comprisinga Mitrecin A polypeptide, a polynucleotide encoding a Mitrecin Apolypeptide, or a Mitrecin A encoding vector. In a particularembodiment, the composition is a food preservative, a dietarysupplement, a topical cream or lotion, a drink, a disinfectant, anantiseptic, a pill, a gargle, a wipe, or a chewing gum.

The invention also includes a method of killing or inhibiting growth ofone or more bacteria comprising contacting the bacteria with aneffective amount of a Mitrecin A polypeptide, a polynucleotide encodingthe Mitrecin A polypeptide, a vector comprising the polynucleotide, ahost cell comprising the vector, or a composition comprising thepolypeptide, the polynucleotide, the vector, or the host cell of thepresent invention, wherein the polypeptide, the polynucleotide, thevector, the host cell, or the composition kills or inhibits growth ofone or more bacteria. In one embodiment, the one or more bacteria are ina food system and are capable of spoiling the food system. In anotherembodiment, the one or more bacteria induce a disease or disorder in aplant or in an animal. For example, by contacting a Mitrecin Apolypeptide, a polynucleotide encoding the Mitrecin A polypeptide, avector comprising the polynucleotide, a host cell comprising the vector,or a composition comprising the polypeptide, the polynucleotide, thevector, or the host cell of the present invention with the one or morebacteria, the polypeptide, the polynucleotide, the vector, the hostcell, or the composition prevents spoilage of the food system for aperiod longer than a food system having no contact with the polypeptide,the polynucleotide, the vector, the host cell, or the composition.

In some embodiments, the invention includes a method of preventing,ameliorating, or treating a disease or disorder in a plant comprisingcontacting the plant with an effective amount of a polypeptide, apolynucleotide encoding the Mitrecin A polypeptide, a vector comprisingthe polynucleotide, a host cell comprising the vector, or a compositioncomprising the polypeptide, the polynucleotide, the vector, or the hostcell of the present invention prevents, ameliorates, or treats thedisease or disorder in the plant. The disease or disorder in the plantcan induce growth inhibition or retardation, less fruit formation, or ahigher death rate of the plant compared to a plant without the diseaseor disorder. In one aspect, the plant is an agricultural crop. Inanother aspect, the contacting is spraying a Mitrecin A polypeptide, apolynucleotide encoding the Mitrecin A polypeptide, a vector comprisingthe polynucleotide, a host cell comprising the vector, or a compositioncomprising the polypeptide, the polynucleotide, the vector, or the hostcell of the present invention on the plant or adding the polypeptide,the polynucleotide, the vector, the host cell, or the composition in thesoil around the plant.

In other embodiments, the invention includes a method of preventing,ameliorating, or treating a disease or disorder in an animal comprisingcontacting the animal with an effective amount of a Mitrecin Apolypeptide, a polynucleotide encoding the Mitrecin A polypeptide, avector comprising the polynucleotide, a host cell comprising the vector,or a composition comprising the polypeptide, the polynucleotide, thevector, or the host cell of the present invention prevents, ameliorates,or treats the disease or disorder in the animal. For example, thecontacting is ingesting of the Mitrecin A polypeptide, thepolynucleotide, the vector, the host cell, or the composition by theanimal or application of the polypeptide, the polynucleotide, thevector, the host cell, or the composition on a skin of the animal. Inone aspect, the animal is a non-human animal. In another aspect, theanimal is human.

In yet other embodiments, the invention is directed to a method ofdetermining pathogenic bacteria in a sample comprising contacting thesample with an effective amount of a Mitrecin A polypeptide, apolynucleotide encoding a Mitrecin A polypeptide, a vector comprisingthe polynucleotide, a host cell comprising the vector, or a compositioncomprising the polypeptide. The method can further comprise measuringkilling or growth inhibition of the pathogenic bacteria in the sample.In one example, the sample can be an environmental sample. In anotherexample, the sample is a tissue of an animal or a plant.

The invention also includes a method of producing a polypeptide whichkills or inhibits growth of one or more bacteria, the method comprisingculturing a host cell comprising a polynucleotide encoding a Mitrecin Apolypeptide of the invention under suitable conditions to express theMitrecin A polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Bacteriocin activity of Streptomyces sp. strain 212. Zymogramanalysis of Streptomyces sp. strain 212 growth supernatant was used tomeasure its bacteriocin activity against Y. pseudotuberculosisimpregnated in a SDS PAGE gel substrate.

FIG. 2. Neighbor-joining cladogram based on nearly complete 16S rRNAgene sequences showing the phylogenetic relationship betweenStreptomyces sp. strain 212 and other Streptomyces species. Percentagesof bootstrap support (>50%) based on 1000 replications are shown at thenodes.

FIG. 3A-3B. FIG. 3A: Stepwise purification of a Mitrecin A 6-histidinefusion protein as shown by SDS-PAGE separation. The lane labeled“Protein Standard” contains molecular mass standards. The lane labeled“Cell Lysate” contains whole cell lysate of the heterologous expressionhost E. coli BL21 (DE3)pLysS. The lane labeled “Affinity PurifiedFraction” contains metal affinity purified Mitrecin A fusion protein.Finally, the lane labeled “SEC Purified Fraction” contains Mitrecin A aspurified by size exclusion chromatography. FIG. 3B: Western BlotAnalysis against the 6x-His tag of purified Mitrecin A fusion protein.Arrows indicate purified Mitrecin A.

FIG. 4A-4C. Factors affecting Mitrecin A activity. Using a quantitativedye-release assay, residual bacteriolytic activity of Mitrecin A (1 μg)was measured (FIG. 4A) at various temp conditions after various30-minute temperature treatments, (FIG. 4B) various pH concentrations,and (FIG. 4C) various saline concentrations.

FIG. 5. Slide diffusion assay measuring the bacteriolytic effects ofMitrecin A on indicator organisms. Right well contains 1 μg of MitrecinA suspended in 10 μl Tris-HCl 50 mM NaCl buffer. Left well containsbuffer with no enzyme. The slide was custom constructed with an overlayof agarose containing Vibrio cholerae cells. Slides were incubated at37° C.

FIG. 6. Bacteriolytic effects of various concentrations of Mitrecin Aagainst Yersinia pseudotuberculosis. Viable cell count assays wereconducted after 16 h incubation at 37° C. in the presence of enzyme.

FIG. 7. Conserved Regions of the catalytic domain of Mitrecin A. TheC-terminus of Mitrecin A (amino acids 54 to 127 of SEQ ID NO: 2) wasaligned with the catalytic domains of closely related zincmetallohydrolases within the C-terminal VanY peptidase superfamily usingClustalW Multiple sequence alignment tool of BioEdit Sequence AlignmentEditor. The conserved amino acid residues and motifs are shaded.Histidine (H) (amino acid residue 63 corresponding to SEQ ID NO: 2),aspartic acid (D) (amino acid residue 70 corresponding to SEQ ID NO: 2),and histidine (H) (amino acid residue 118 corresponding to SEQ ID NO: 2)residues inferred by analysis in the MEROPS database that may benecessary for interaction with the zinc ion within the binding pocket ofthe catalytic site are enclosed in boxes. The active site residue(aspartic acid (D) at amino acid residue 115 corresponding to SEQ ID NO:2) as determined by analysis in MEROPS is also shaded. The catalyticdomain of Mitrecin A contains three conserved regions (Conserved Region1, Conserved Region 2, and Conserved Region 3). Conserved Region 1comprises amino acids 54 to 73 of SEQ ID NO: 2. Conserved Region 2comprises amino acids 81 to 90 of SEQ ID NO: 2. Conserved Region 3comprises amino acids 106 to 121 of SEQ ID NO: 2. C-terminus domains ofthe sequences with similarity to Mitrecin A identified in MEROPS Blastsearches and subsequently aligned against the Mitrecin A C-terminusinclude phage endolysin from Brevundimonas subvibrioides ATCC 15264(Endolysin Bsub; NCBI YP_003819570.1, SEQ ID NO: 6), hypothetic proteinBBAL3 176 from Brevundimonas sp. BAL3 (Protein BBAL3 176; NCBIYP_002588038.1, SEQ ID NO: 7), endolysin from Shigella dysenteriaestrain 1012 (Endolysin Sdys; NCBI ZP_03065132.1, SEQ ID NO: 8), putativephage endolysin from Escherichia coli strain SE11 (Endolysin Ecoli SE11;NCBI YP_002293134.1, SEQ ID NO: 9), putative endolysin fromEnterobacteria phase Phi27 (Endolysin Phi27; NCBI NP_543082.1, SEQ IDNIO: 10), and L-alanyl-D-glutamate peptidase from Micavibrioaeruginosavorus strain ARL-13 (Peptidase Maer; NCBI AEP08879.1, SEQ IDNO: 11).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to polypeptides and polynucleotidesthat are capable of killing or inhibiting growth of one or more targetbacterium and their uses in killing or inhibiting growth of one or moretarget bacterium.

Methods of making and using the present invention include allconventional techniques of molecular biology, microbiology, andrecombinant DNA technology. Such techniques are set forth in theliterature including but not limited to e.g. Sambrook Molecular Cloning;A Laboratory Manual, Second Edition (1989) and Third Edition (2001);Genetic Engineering: Principles and Methods, Volumes 1-25 (J. K. Setlowed, 1988); DNA Cloning, Volumes I and II (D. N Glover ed. 1985);Oligonucleotide Synthesis (M. J. Gait ed, 1984); Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription andTranslation (B. D. Hames & S. J. Higgins eds. 1984); Immobilized Cellsand Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to MolecularCloning (1984); the Methods in Enzymology series (Academic Press, Inc.),especially volumes 154 & 155; Scopes, (1987) Protein Purification:Principles and Practice, Second Edition (Springer-Verlag, N.Y.), andHandbook of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C.Blackwell eds 1986). (Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press andAusubel et al. Eds. (1997) Current Protocols in Molecular Biology, JohnWiley & Sons, Inc.).

Definitions

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “a polypeptide,” is understood to representone or more polypeptides. As such, the terms “a” (or “an”), “one ormore,” and “at least one” can be used interchangeably herein.

The term “nucleic acid,” “nucleotide,” “nucleic acid fragment,” or“nucleotide fragment” refers to any one or more nucleic acid segments,e.g., DNA or RNA fragments, present in a polynucleotide or construct.Two or more nucleic acids of the present invention can be present in asingle polynucleotide construct, e.g., on a single plasmid, or inseparate (non-identical) polynucleotide constructs, e.g., on separateplasmids. Furthermore, any nucleic acid or nucleic acid fragment mayencode a single polypeptide, e.g., a single antigen, cytokine, orregulatory polypeptide, or may encode more than one polypeptide, e.g., anucleic acid may encode two or more polypeptides. In addition, a nucleicacid may comprise a regulatory element such as a promoter or atranscription terminator, or may encode a specialized element or motifof a polypeptide or protein, such as a secretory signal peptide or afunctional domain.

The term “polynucleotide” is intended to encompass a single nucleic acidor nucleic acid fragment as well as plural nucleic acids or nucleic acidfragments, and refers to an isolated molecule or construct, e.g., avirus genome (e.g., a non-infectious viral genome), messenger RNA(mRNA), plasmid DNA (pDNA), or derivatives of pDNA (e.g., minicircles asdescribed in (Darquet, A-M et al., Gene Therapy 4:1341-1349 (1997))comprising a polynucleotide. A polynucleotide may be provided in linear(e.g., mRNA), circular (e.g., plasmid), or branched form as well asdouble-stranded or single-stranded forms. A polynucleotide may comprisea conventional phosphodiester bond or a non-conventional bond (e.g., anamide bond, such as found in peptide nucleic acids (PNA)).

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and comprisesany chain or chains of two or more amino acids. Thus, as used herein,terms including, but not limited to “peptide,” “dipeptide,”“tripeptide,” “protein,” “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids, are included inthe definition of a “polypeptide,” and the term “polypeptide” may beused instead of, or interchangeably with any of these terms. The termfurther includes polypeptides which have undergone post-translationalmodifications, for example, glycosylation, acetylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, or modification by non-naturally occurring aminoacids.

The terms “fragment” when referring to polypeptides of the presentinvention include any polypeptides which retain at least some of theantimicrobial activity of the original sequence (i.e., SEQ ID NO: 2).Fragments of Mitrecin A polypeptides of the present invention includeproteolytic fragments, deletion fragments and fragments of a Mitrecin Apolypeptides which exhibit increased solubility during expression orpurification. In one example, a Mitrecin A polypeptide fragmentcomprises the catalytic domain of the full-length Mitrecin Apolypeptide.

The term “analog,” “derivative,” or “variant,” as used herein, can beused interchangeably and refers to a polypeptide that differs from therecited polypeptide due to amino acid substitutions, deletions,insertions, and/or modifications. Variants, derivatives, or analogs mayoccur naturally. Non-naturally occurring variants, analogs, orderivatives may be produced using art-known mutagenesis techniques. Inone embodiment, variant, analog, or derivative polypeptides differ froman identified sequence by substitution, deletion or addition of fiveamino acids or fewer. Such variants may generally be identified bymodifying a polypeptide sequence, and evaluating the antigenicproperties of the modified polypeptide using, for example, therepresentative procedures described herein.

Polypeptide variants can exhibit at least about 60-70%, for example 75%,80%, 85%, 90%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% sequence identitywith identified polypeptides. Variant polypeptides may compriseconservative or non-conservative amino acid substitutions, deletions oradditions. Examples include fusion proteins. An analog is another formof a polypeptide of the present invention.

Variants may also, or alternatively, contain other modifications,whereby, for example, a polypeptide may be conjugated or coupled, e.g.,fused to a heterologous polypeptide, e.g., a signal (or leader) sequenceat the N-terminal end of the protein which co-translationally orpost-translationally directs transfer of the protein. The polypeptidemay also be conjugated to a linker or other sequence for ease ofsynthesis, purification or identification of the polypeptide (e.g.,6-His), or to enhance binding of the polypeptide to a solid support. Thepolypeptide may also be conjugated or coupled to other polypeptides fromanother bacterium and/or other viruses to generate a hybrid protein thathas antimicrobial activities to broader spectrum of pathogenicorganisms.

Polypeptides, and fragments, derivatives, analogs, or variants thereofof the present invention can be bacteriocin polypeptides, which are usedto kill or inhibit growth of one or more bacteria. The term“bacteriocin” as used herein refers to a substance that is capable ofkilling or inhibiting growth of one or more target bacteria when thesubstance comes in contact with the one or more bacteria.

As used herein, the term “isolated” means that the polynucleotide orpolypeptide or fragment, variant, or derivative thereof has been removedfrom other biological materials with which it is naturally associated.An example of an isolated polynucleotide is a recombinant polynucleotidecontained in a vector. Further examples of an isolated polynucleotideinclude recombinant polynucleotides maintained in heterologous hostcells or purified (partially or substantially) polynucleotides insolution. Isolated RNA molecules include in vivo or in vitro RNAtranscripts of the polynucleotides of the present invention. Isolatedpolynucleotides or nucleic acids according to the present inventionfurther include such molecules produced synthetically.

As used herein, the term “purified” means that the polynucleotide orpolypeptide or fragment, variant, or derivative thereof is substantiallyfree of other biological material with which it is naturally associated,or free from other biological materials derived, e.g., from arecombinant host cell that has been genetically engineered to expressthe polypeptide of the invention.

The term “sequence identity” as used herein refers to a relationshipbetween two or more polynucleotide sequences or between two or morepolypeptide sequences. When a position in one sequence is occupied bythe same nucleic acid base or amino acid residue in the correspondingposition of the comparator sequence, the sequences are said to be“identical” at that position. The percentage “sequence identity” iscalculated by determining the number of positions at which the identicalnucleic acid base or amino acid residue occurs in both sequences toyield the number of “identical” positions. The number of “identical”positions is then divided by the total number of positions in thecomparison window and multiplied by 100 to yield the percentage of“sequence identity.” Percentage of “sequence identity” is determined bycomparing two optimally aligned sequences over a comparison window(e.g., SEQ ID NO: 2). In order to optimally align sequences forcomparison, the portion of a polynucleotide or polypeptide sequence inthe comparison window may comprise additions or deletions termed gapswhile the reference sequence (e.g. SEQ ID NO: 2) is kept constant. Anoptimal alignment is that alignment which, even with gaps, produces thegreatest possible number of “identical” positions between the referenceand comparator sequences. Percentage “sequence identity” between twosequences can be determined using the version of the program “BLAST 2Sequences” which was available from the National Center forBiotechnology Information as of Sep. 1, 2004, which program incorporatesthe programs BLASTN (for nucleotide sequence comparison) and BLASTP (forpolypeptide sequence comparison), which programs are based on thealgorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA90(12):5873-5877, 1993). When utilizing “BLAST 2 Sequences,” parametersthat were default parameters as of Sep. 1, 2004, can be used for wordsize (3), open gap penalty (11), extension gap penalty (1), gap dropoff(50), expect value (10) and any other required parameter including butnot limited to matrix option.

The term “heterologous” refers to any additional biological componentsthat are not identical with the subject biological component.

As used herein, a “coding region” is a portion of nucleic acid whichconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it may beconsidered to be part of a coding region, but any flanking sequences,for example promoters, ribosome binding sites, transcriptionalterminators, and the like, are outside the coding region.

The term “codon optimization” is defined herein as modifying a nucleicacid sequence for enhanced expression in the cells of the host ofinterest by replacing at least one, more than one, or a significantnumber, of codons of the native sequence with codons that are morefrequently or most frequently used in the genes of that host. Variousspecies exhibit particular bias for certain codons of a particular aminoacid.

An “effective amount” is the amount of which is sufficient to kill orinhibit growth of one or more bacteria or to detect or measure presenceof one or more pathogenic bacteria. An amount is effective, for example,when its addition to or contact with the target bacteria results in thekilling of the bacteria or reduction of the growth of the bacteria. Thisamount varies depending upon the condition of the target bacteria to bekilled or inhibited, the number of the target bacteria, the family orspecies of the bacteria, the capacity of the food system or object thatmay contain the bacteria, the differences of the plant or animal to beprotected, the degree of protection desired, and other relevant factors.It is expected that the effective amount will fall in a relatively broadrange that can be determined through routine trials. Typically a singledose is from about 1 μg to 100 mg/ml of purified polypeptide.

The term “contacting” as used herein includes applying a Mitrecin Apolypeptide to one or more bacteria or to an environment where bacteriaare expected to grow, e.g., a hospital surface or water where bacteriaare thought to be present. In one embodiment, “contacting” means mixinga Mitrecin A polypeptide with one or more bacteria in a container or ona surface on which one or more bacteria grow or may grow. In anotherembodiment, “contacting” means administering or applying a Mitrecin Apolypeptide to a subject in need thereof that comes in contact with oris expected to come in contact with one or more bacteria.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent or slow down(lessen) the targeted pathologic condition or disorder, which is acondition or disorder caused by pathological bacteria, for example,Gram-positive pathological bacteria, more specifically staphylococci,and more specifically Staphylococcus aureus. Those in need of treatmentinclude those already with the disorder as well as those prone to havethe disorder or those for whom the disorder is to be prevented.

The term “non-human” is meant any animal other than human, particularlya livestock, for whom the protection from the target bacteria isdesired. In one embodiment, the non-human animal is a non-human mammal.Mammalian subjects include, but are not limited to, domestic animals,farm animals, zoo animals such as bears, sport animals, pet animals suchas dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears,cows; primates such as apes, monkeys, orangutans, and chimpanzees;canids such as dogs and wolves; felids such as cats, lions, and tigers;equids such as horses, donkeys, and zebras; food animals such as cows,pigs, and sheep; ungulates such as deer and giraffes; rodents such asmice, rats, hamsters and guinea pigs; and so on. In another embodiment,the animal is a human.

The term “animal” is intended to encompass a singular “animal” as wellas plural “animals” and comprises mammals and birds, as well as fish,reptiles, and amphibians. The term animal also encompasses modelanimals, e.g., disease model animals. In some embodiments, the termanimal includes valuable animals, either economically or otherwise,e.g., economically important breeding stock, racing animals, showanimals, heirloom animals, rare or endangered animals, or companionanimals. In particular, the mammal can be a human subject, a food animalor a companion animal.

Polypeptides

The present invention is drawn to a bacteriocin polypeptide or peptidereferred to as a Mitrecin A polypeptide. The polypeptide of theinvention is useful in killing or inhibiting growth of one or moretarget microorganisms and/or detecting the presence of one or moremicroorganisms. The polypeptide of the invention can be eitherbactericidal, bacteriostatic, or both. The polypeptide of the inventionused as a bactericidal agent kills (e.g., is capable of killing) ordestroys one or more bacteria. The polypeptide can thus be used asdisinfectants, antiseptics, or antibiotics. The polypeptide of theinvention used as a bacteriostatic agent inhibits growth of one or morebacteria from reproducing, while not necessarily harming them otherwise.Upon removal of the bacteriostatic agent, the bacteria can start to growagain.

In one embodiment, the invention is an isolated polypeptide comprisingan amino acid sequence at least about 90% identical to SEQ ID NO: 2 or afragment thereof, wherein the polypeptide kills or inhibits growth ofone or more bacteria. The invention also includes a polypeptidecomprising a functional fragment of SEQ ID NO: 2, wherein thepolypeptide kills or inhibits growth of one or more bacteria. Oneexample of the functional fragments of the full-length Mitrecin Apolypeptide (i.e., SEQ ID NO: 2) is a polypeptide comprising a catalyticdomain of the Mitrecin A polypeptide. In another embodiment, theinvention is an isolated polypeptide consisting essentially of orconsisting of an amino acid sequence at least about 90% identical to SEQID NO: 2 or a fragment thereof, wherein the polypeptide kills orinhibits growth of one or more bacteria. In one aspect, the polypeptideof the invention is bactericidal. In another aspect, the polypeptide ofthe invention is bacteriostatic. In some aspects, the polypeptide of theinvention is both bactericidal and bacteriostatic. In some embodiments,the amino acid sequence in the polypeptide of the invention can be atleast about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 2. In certain embodiments, the amino acid sequence that is atleast about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 2 comprises histidine (H) at amino acid residue 63corresponding to SEQ ID NO: 2, aspartic acid (D) at amino acid residue70 corresponding to SEQ ID NO: 2, aspartic acid (D) at amino acidresidue 115 corresponding to SEQ ID NO: 2, histidine (H) at amino acidresidue 118 corresponding to SEQ ID NO: 2, or any combinations thereof.In other embodiments, the amino acid sequence that is at least about91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2comprises histidine (H) at amino acid residue 63 corresponding to SEQ IDNO: 2, aspartic acid (D) at amino acid residue 70 corresponding to SEQID NO: 2, aspartic acid (D) at amino acid residue 115 corresponding toSEQ ID NO: 2, and histidine (H) at amino acid residue 118 correspondingto SEQ ID NO: 2. In a particular embodiment, the amino acid sequence inthe isolated polypeptide is SEQ ID NO: 2. In still other embodiments,the polypeptide of the invention comprises an amino acid sequenceencoded by a nucleotide sequence at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identical to SEQ ID NO: 1. In a particular embodiment,the polypeptide comprises an amino acid sequence encoded by SEQ ID NO:2. An amino acid sequence of a Mitrecin A polypeptide and a nucleotidesequence encoding a Mitrecin A polypeptide are listed in Table 1.

TABLE 1 Amino Acid and Nucleotide Sequences of Mitrecin A. IdentifierSequence Amino acids of MSFGLSQRSR ERLKGVHPDL VAVVEAAIRL Mitrecin ATPVDFMITEG LRTPARQAEL VRAGASRTLN (SEQ ID NO: 2)SRHLTGHAVD VAAWIDGEVR WDWPLYPRIA EAFKAAAKDR DVALIWGGDW PRLRDGPHFELDRRGYP Nucleic acids atgagcttcggtctgtcgcaacgctcgcgcga encodinggcggttgaagggcgttcatcctgatctggtcg Mitrecin Accgtggtcgaggcggcgatccgcctgacgccg (SEQ ID NO: 1)gtggacttcatgatcacagagggattgcgcac gcccgcgcgccaggcggaactggtccgcgcgggggccagccggacgctgaactcgcgccacttg acgggccatgcggtggatgtcgccgcatggatcgacggcgaggtgcgctgggactggccgctgt acccccgcatcgccgaggcgttcaaggcggcggcgaaggaccgggatgtggctctgatctgggg cggcgactggccgcgcctgcgcgacggaccgcatttcgaactggatcggaggggctacccatga

Peptide and polypeptide sequences defined herein are represented byone-letter symbols for amino acid residues as follows: A (alanine); R(arginine); N (asparagine); D (aspartic acid); C (cysteine); Q(glutamine); E (glutamic acid); G (glycine); H (histidine); I(isoleucine); L (leucine); K (lysine); M (methionine); F(phenylalanine); P (proline); S (serine); T (threonine); W (tryptophan);Y (tyrosine); and V (valine).

Polynucleotides or nucleic acid sequences defined herein are representedby one-letter symbols for the bases as follows: A (adenine) C (cytosine)G (guanine) T (thymine) U (uracil) M (A or C) R (A or G) W (A or T/U);S(C or G); Y (C or T/U); K (G or T/U); V (A or C or G; not T/U); H (A orC or T/U; not G); D (A or G or T/U; not C); B (C or G or T/U; not A); N(A or C or G or T/U) or (unknown).

Also provided is an isolated polypeptide comprising an amino acidsequence at least about 90% identical to SEQ ID NO: 2 or a fragmentthereof, wherein the amino acid sequence has a molecular weight of about14 kDa as visualized by SDS-PAGE. In one embodiment, the polypeptide iscapable of killing or inhibiting the growth of Gram-positive orGram-negative target bacteria.

The present invention also includes an isolated polypeptide comprisingfull-length or mature Mitrecin A polypeptides as well as an isolatedpolypeptide comprising analogs, variants, derivatives, or fragments ofthe Mitrecin A polypeptides described herein. In one embodiment, theanalogs, variants, derivatives, or fragments of the Mitrecin Apolypeptide are capable of killing or inhibiting growth of one or morebacteria. In another embodiment, the analogs, variants, derivatives, orfragments of the Mitrecin A polypeptide are capable of specificallybinding to an antibody raised against a polypeptide consisting of SEQ IDNO: 2 or a fragment thereof or an antibody raised against a polypeptideconsisting of amino acids 54 to 121 of SEQ ID NO: 2. In otherembodiments, the analogs, variants, derivatives, or fragments of theMitrecin A polypeptide are capable of interacting with a zinc ion.

In one embodiment, a Mitrecin A polypeptide comprises, consistsessentially thereof, or consists of a catalytic domain of the Mitrecin Apolypeptide. The catalytic domain of the Mitrecin A polypeptide hasconserved regions and key residues involved in facilitating thecatalytically active site of the zinc metalloenzyme. The catalyticdomain of the Mitrecin A polypeptide can comprise amino acids 54 to 121of SEQ ID NO: 2. In another embodiment, a Mitrecin A polypeptidecomprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, or 100% identical to amino acids 54 to 121 of SEQ ID NO: 2. Asone of skill in the art will appreciate, the beginning and endingresidues of the domains may vary depending upon the computer modelingprogram used or the method used for determining the domains.

The catalytic domain of Mitrecin A comprises three highly conservedregions. The three conserved regions of the catalytic domain are shownin Table 2.

TABLE 2 Mitrecin A Conserved Regions Conserved region 1 Conserved region2 Conserved region 3 Amino acids Amino acids Amino acids 106 to 121 of54 to 73 of 81 to 90 of SEQ ID NO: 2 SEQ ID NO: 2 SEQ ID NO: 2

In one embodiment, an isolated polypeptide of the invention comprisesConserved Region 1 (e.g., amino acids 54 to 73 of SEQ ID NO: 2),Conserved Region 2 (e.g., amino acids 81 to 90 of SEQ ID NO: 2), orConserved Region 3 (e.g., amino acids 106 to 121 of SEQ ID NO: 2). Inanother embodiment, an isolated polypeptide of the invention comprisesConserved Region 1 and Conserved Region 2, Conserved Region 2 andConserved Region 3, Conserved Region 1 and Conserved Region 3, orConserved Region 1, Conserved Region 2, and Conserved Region 3. In otherembodiments, an isolated polypeptide of the invention comprises aminoacids 54 to 90 of SEQ ID NO: 2, amino acids 81 to 121 of SEQ ID NO: 2,or amino acids 54 to 121 of SEQ ID NO: 2. In some embodiments, anisolated polypeptide of the invention comprises amino acids 54 to 127 ofSEQ ID NO: 2. The isolated polypeptides of the invention comprising oneor more Conserved Regions are capable of killing or inhibiting growth ofone or more bacteria, specifically binding to an antibody raised againsta polypeptide consisting of SEQ ID NO: 2 or a fragment thereof or apolypeptide consisting of amino acids 54 to 121 of SEQ ID NO: 2, orinteracting with a zinc ion.

In certain embodiments, the present invention includes a polypeptidecomprising at least two catalytic domains, at least three catalyticdomains, at least four catalytic domains, or at least five catalyticdomains derived from the Mitrecin A polypeptide.

Examples of Gram-positive target bacteria that can be killed orinhibited by the polypeptides of the present invention include, but arenot limited to, Streptomyces avermitilis, Streptomyces coelicolor,Streptomyces lividans, Streptomyces venezuelae, Nocardia salmonicida,Nocardia vaccinii, Rhodococcus marinonascens, Bacillus megaterium,Bacillus subtilis, Bacillus cereus, Enterococcus faecalis, Micrococcusluteus, Staphylococcus aureus, Streptococcus sp., Streptococcuspyogenes, Listeria monocytogenes, Clostridium perfringens, Clostridiumbotulinum, Lactococcus cremoris, Lactobacillus sp., and Leuconostoc sp.Thus, a Gram-positive target bacterium can be selected from the groupconsisting of Streptomyces avermitilis, Streptomyces coelicolor,Streptomyces lividans, Streptomyces venezuelae, Nocardia salmonicida,Nocardia vaccinii, Rhodococcus marinonascens, Bacillus megaterium,Bacillus subtilis, Bacillus cereus, Enterococcus faecalis, Micrococcusluteus, Staphylococcus aureus, Streptococcus sp., Streptococcuspyogenes, Listeria monocytogenes, Clostridium perfringens, Clostridiumbotulinum, Lactococcus cremoris, Lactobacillus sp., and Leuconostoc sp.

Examples of Gram-negative target bacteria that can be killed orinhibited by the polypeptides of the present invention include, but arenot limited to, Escherichia coli, Klebsiella pneumoniae, Salmonellatyphimurium, Salmonella enterica, Campylobacter jejuni, Yersiniaenterocolitica, Yersinia pseudotuberculosis, Vibrio cholerae, Vibrioparahaemolyticus, Vibrio vulnificus, Aeromonas hydrophila, Plesiomonasshigelloides, Shigella sonnei, Shigella flexneri, Enterobacteraerogenes, Flavobacterium sp., Acinetobacter sp., and Proteus sp. Thus,a Gram-negative target bacterium can be selected from the groupconsisting of Escherichia coli, Klebsiella pneumoniae, Salmonellatyphimurium, Salmonella enteritidis, Campylobacter jejuni, Yersiniaenterocolitica, Yersinia pseudotuberculosis, Vibrio cholerae, Vibrioparahaemolyticus, Vibrio vulnificus, Aeromonas hydrophila, Plesiomonasshigelloides, Shigella sonnei, Shigella flexneri, Enterobacteraerogenes, Flavobacterium sp., Acinetobacter sp., and Proteus sp.

In some embodiments, the target bacterium is Vibrio sp., Salmonella sp.,Yersinia sp., Shigella sp. Aeromonas sp., or Bacillus sp. In aparticular embodiment, the target bacterium is Vibrio cholerae,Salmonella enterica, Yersinia pseudotuberculosis, Shigella sonnei,Aeromonas hydrophila, or Bacillus subtilis.

In yet other embodiments, the target bacterium does not kill or inhibitgrowth of the cell line comprising a polynucleotide encoding a MitrecinA polypeptide.

In other embodiments, the present invention includes a fusionpolypeptide fused with a heterologous moiety. The heterologous moietyfused to a Mitrecin A polypeptide or a fragment, variant, analog, orderivative thereof can be a heterologous polypeptide, a non-polypeptidemoiety, or both. The heterologous polypeptide can be translated fromvarious heterologous nucleic acids. Various heterologous polypeptidescan be used, and can be selected from the group consisting of an N- orC-terminal peptide imparting stabilization, secretion, or simplifiedpurification, i.e., His-tag (SEQ ID NO: 3), ubiquitin tag, NusA tag,chitin binding domain, ompT, ompA, pelB, DsbA, DsbC, c-myc, KSI,polyaspartic acid, (Ala-Trp-Trp-Pro)n, polyphenyalanine, polycysteine,polyarginine, B-tag, HSB-tag, green fluorescent protein (GFP),hemagglutinin influenza virus (HAI), calmodulin binding protein (CBP),galactose-binding protein, maltose binding protein (MBP), cellulosebinding domains (CBD's), dihydrofolate reductase (DHFR),glutathione-S-transferase (GST), streptococcal protein G, staphylococcalprotein A, T7gene10, avidin/streptavidin/Strep-tag, trpE,chloramphenicol acetyltransferase, lacZ (β-Galactosidase), His-patchthioredoxin, thioredoxin, FLAG™ peptide (Sigma-Aldrich), S-tag, andT7-tag. See e.g., Stevens, R. C., Structure, 8:R177-R185 (2000). Theheterologous polypeptides can further include any pre- and/orpro-sequences that facilitate the transport, translocations, processingand/or purification of the Mitrecin A polypeptide from a host cell. Insome embodiments, the heterologous moiety comprises one or morebacteriocins (either native proteins or variants, fragments, orderivatives thereof, e.g., nisin).

Optionally, a Mitrecin A polypeptide fused with a heterologouspolypeptide or other bacteriocin polypeptide can include a peptidelinker sequence joining sequences that comprise two or more sequenceshaving antimicrobial activities. Suitable peptide linker sequences maybe chosen based on their ability to adopt a flexible, extendedconformation, or a secondary structure that could interact with joinedsequences, or based on their ability to increase overall solubility ofthe fusion polypeptide, or based on their lack of electrostatic orwater-interaction effects that influence joined sequences. A linkersequence, (GGGS)_(n) (SEQ ID NO: 4), where n can be 0, 1, 2, 3, 4, ormore, can be added at N-terminal and/or C-terminal of the Mitrecin Apolypeptide. Alternative linkers such as PEAPTDPEAPTD (SEQ ID NO: 5) canalso be employed in place of GGGS linker (Kim et al., J Pharmacol. Exp.Ther., 2010, 334, 682-692).

In some embodiments, the polypeptides of the present invention areisolated. No particular level of purification is required. Recombinantlyproduced endolysin polypeptides and proteins expressed in host cells areconsidered isolated for purposes of the invention, as are native orrecombinant endolysin polypeptides which have been separated,fractionated, or partially or substantially purified by any suitabletechnique, including by filtration, chromatography, centrifugation, andthe like.

In other embodiments, the polypeptides of the invention are at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least100% purified.

In certain embodiments, the invention provides an antibody specificallybinds to a Mitrecin A polypeptide. An anti-Mitrecin A antibody can be amonoclonal antibody or a polyclonal antibody. In further embodiments,the invention also includes an anti-serum raised against a polypeptideconsisting of SEQ ID NO: 2 or a fragment thereof. The anti-Mitrecin Aantibody or antiserum can be used to detect or purify a Mitrecin Apolypeptide.

Polynucleotides

The present invention also includes a polynucleotide comprising anucleic acid encoding a full-length Mitrecin A polypeptide or, fragment,analog, derivative, or variant thereof or a complementary sequencethereof.

Polynucleotide sequences encoding Mitrecin A polypeptides may be clonedfrom genomic DNA from Streptomyces sp. 212 by a variety of techniquesknown in the art. For instance, Streptomyces sp. 212 can be cultured asa cell culture as described in the art. Streptomyces sp. 212 genomic DNAcan be isolated, for example, as described in the art. Full lengthMitrecin A genes may be cloned, for example, by PCR amplification.Suitable primers are described herein, or may be designed usingtechniques well known in the art. An oligonucleotide forward primer,encoding a known N-terminal sequence of Mitrecin A, may be used togetherwith an oligonucleotide reverse primer, which comprises the reversecomplement of a known C-terminal sequence. Suitable primers may includea synthetic restriction enzyme cleavage site which is not found withinthe Mitrecin A coding sequence, to facilitate manipulation of the PCRamplification clone. Alternatively, degenerate primers may be designedwhich permit amplification of corresponding genes despite a high degreeof sequence variability. PCR conditions can also be modified to allowfor a greater degree of sequence similarity, as is well known to thoseskilled in the art.

Included within the scope of the invention are polynucleotides at leastabout 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to polynucleotides encoding Mitrecin A polypeptides,fragments, derivatives, or variants thereof. In one aspect, the MitrecinA polypeptides, fragments, derivatives, or variants thereof are capableof killing or inhibiting growth of one or more bacteria or are capableof specifically binding to an antibody raised against a polypeptideconsisting of SEQ ID NO: 2 or a fragment thereof or a polypeptideconsisting of amino acids 54 to 121 of SEQ ID NO: 2. In another aspect,the Mitrecin A polypeptides, fragments, derivatives, or variants thereofare capable of interacting with a zinc ion. Variants commonly occur withmany genes of simple, unicellular organisms. Examples of variants can befound among natural variants. Another example of variants can be foundwithin strains.

In one embodiment, the present invention includes a polynucleotidecomprising, consisting essentially of, or consisting of a nucleic acidencoding an amino acid sequence at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% identical to amino acids 54 to 127 of SEQ ID NO:2. In another embodiment, a polynucleotide encoding a Mitrecin Apolypeptide comprises a nucleotide sequence encoding an amino acidsequence comprising (1) Conserved Region 1, (2) Conserved Region 2, (3)Conserved Region 3, (4) Conserved Region 1 and Conserved Region 2, (5)Conserved Region 1 and Conserved Region 3, (6) Conserved Region 2 andConserved Region 3, or (7) Conserved Region 1, Conserved Region 2, andConserved Region 3. In other embodiments, a polynucleotide of theinvention comprises a nucleotide sequence encoding amino acids 54 to 90of SEQ ID NO: 2, amino acids 81 to 121 of SEQ ID NO: 2, amino acids 54to 121 of SEQ ID NO: 2, or amino acids 54 to 127 of SEQ ID NO: 2.

In other embodiments, a polynucleotide of the invention comprises anucleotide sequence encoding an amino acid sequence at least about 90%,95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2, wherein theamino acid sequence is capable of killing or inhibiting growth of one ormore bacteria or is capable of specifically binding to an antibodyraised against a polypeptide consisting of SEQ ID NO: 2 or a fragmentthereof or a polypeptide consisting of amino acids 54 to 121 of SEQ IDNO: 2. In certain embodiments, a polynucleotide of the inventioncomprises a nucleotide sequence at least 70%, 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, wherein theamino acid sequence encoded by the nucleotide sequence is capable ofkilling or inhibiting growth of one or more bacteria or is capable ofspecifically binding to an antibody raised against a polypeptideconsisting of SEQ ID NO: 2 or a fragment thereof or a polypeptideconsisting of amino acids 54 to 121 of SEQ ID NO: 2. In otherembodiments, the nucleotide sequence comprises nucleic acids encodinghistidine (H) at amino acid residue 63 corresponding to SEQ ID NO: 2,aspartic acid (D) at amino acid residue 70 corresponding to SEQ ID NO:2, aspartic acid (D) at amino acid residue 115 corresponding to SEQ IDNO: 2, histidine (H) at amino acid residue 118 corresponding to SEQ IDNO: 2, or any combinations thereof. In other embodiments, the nucleotidesequence that is at least about 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 comprises nucleic acidsencoding histidine (H) at amino acid residue 63 corresponding to SEQ IDNO: 2, aspartic acid (D) at amino acid residue 70 corresponding to SEQID NO: 2, aspartic acid (D) at amino acid residue 115 corresponding toSEQ ID NO: 2, and histidine (H) at amino acid residue 118 correspondingto SEQ ID NO: 2. In a particular embodiment, the nucleotide sequence inthe isolated polynucleotide is SEQ ID NO: 1.

In some embodiments of the present invention the polynucleotide isisolated. In other embodiments, the polynucleotide is purified. Forexample, a purified polypeptide of the present invention includes apolypeptide that is at least 70-100% pure, i.e., a polypeptide which ispresent in a composition wherein the polypeptide constitutes 70-100% byweight of the total composition. In some embodiments, the purifiedpolypeptide of the present invention is 75%-99% by weight pure, 80%-99%by weight pure, 90-99% by weight pure, or 95% to 99% by weight pure. Anexample of an isolated polynucleotide is a recombinant polynucleotidecontained in a vector. Further examples of an isolated polynucleotideinclude recombinant polynucleotides maintained in heterologous hostcells or purified (partially or substantially) polynucleotides insolution. Isolated RNA molecules include in vivo or in vitro RNAtranscripts of the polynucleotides of the present invention. Isolatedpolynucleotides or nucleic acids according to the present inventionfurther include such molecules produced synthetically. The relativedegree of purity of a polynucleotide or polypeptide of the invention iseasily determined by well-known methods.

In some embodiments, the present invention is directed to apolynucleotide encoding Mitrecin A further comprising a heterologousnucleic acid. The heterologous nucleic acid can, in some embodiments,encode a heterologous polypeptide fused to a Mitrecin A polypeptide. Thecomponents may be host cells, genes, or regulatory regions, such aspromoters. The heterologous components can function together, as when apromoter heterologous to a gene is operably linked to the gene. Anexample would be two epitopes from the same or different proteins whichhave been assembled in a single protein. Another example includes apolynucleotide which encodes an Fc portion of an antibody linked to aMitrecin A polypeptide of the invention or a fragment thereof. A furtherexample is a full-length or mature Mitrecin A polypeptide fused to a 6histidine tag, i.e., SEQ ID NO: 3. The present invention includes apolynucleotide comprising a nucleic acid which encodes an amino acidsequence at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identicalto SEQ ID NO: 2 without a signal sequence and a heterologouspolypeptide, e.g., SEQ ID NO: 3, wherein said polypeptide is capable ofkilling or inhibiting growth of one or more bacteria.

Various heterologous nucleic acids can be used to encode theirrespective heterologous polypeptides. In some embodiments, theheterologous polypeptide is fused to the polypeptide of the presentinvention. Examples of the heterologous polypeptide that can be encodedby the heterologous polynucleotides are disclosed elsewhere herein.

Also included within the scope of the invention are geneticallyengineered polynucleotides encoding Mitrecin A variants. Modificationsof nucleic acids encoding Mitrecin A polypeptides can readily beaccomplished by those skilled in the art, for example, byoligonucleotide-directed site-specific mutagenesis of a polynucleotidecoding for a Mitrecin A polypeptide. Such modified polypeptide can beencoded by a codon optimized nucleotide sequence. Such modificationsimpart one or more amino acid substitutions, insertions, deletions,and/or modifications to expressed Mitrecin A polypeptides includingfragments, variants, and derivatives. Such modifications may enhance theantimicrobial activity of Mitrecin A polypeptides, for example. Suchmodification may enhance solubility of the polypeptides. Alternatively,such modifications may have no effect.

As appreciated by one of ordinary skill in the art, various nucleic acidcoding regions will encode the same polypeptide due to the redundancy ofthe genetic code. Deviations in the nucleotide sequence that comprisethe codons encoding the amino acids of any polypeptide chain allow forvariations in the sequence coding for the gene. Since each codonconsists of three nucleotides, and the nucleotides comprising DNA arerestricted to four specific bases, there are 64 possible combinations ofnucleotides, 61 of which encode amino acids (the remaining three codonsencode signals ending translation). The “genetic code” which shows whichcodons encode which amino acids is reproduced herein as Table 3. As aresult, many amino acids are designated by more than one codon. Forexample, the amino acids alanine and proline are coded for by fourtriplets, serine and arginine by six, whereas tryptophan and methionineare coded by just one triplet. This degeneracy allows for DNA basecomposition to vary over a wide range without altering the amino acidsequence of the polypeptides encoded by the DNA.

TABLE 3 The Standard Genetic Code T C A G T TTT Phe (F) TCT Ser (S) TATTyr (Y) TGT Cys (C) TTC Phe (F) TCC Ser (S) TAC Tyr (Y) TGC TTA Leu (L)TCA Ser (S) TAA Ter TGA Ter TTG Leu (L) TCG Ser (S) TAG Ter TGG Trp (W)C CTT Leu (L) CCT Pro (P) CAT His (H) CGT Arg (R) CTC Leu (L) CCC Pro(P) CAC His (H) CGC Arg (R) CTA Leu (L) CCA Pro (P) CAA Gln (Q) CGA Arg(R) CTG Leu (L) CCG Pro (P) CAG Gln (Q) CGG Arg (R) A ATT Ile (I) ACTThr (T) AAT Asn (N) AGT Ser (S) ATC Ile (I) ACC Thr (T) AAC Asn (N) AGCSer (S) ATA Ile (I) ACA Thr (T) AAA Lys (K) AGA Arg (R) ATG Met (M) ACGThr (T) AAG Lys (K) AGG Arg (R) G GTT Val (V) GCT Ala (A) GAT Asp (D)GGT Gly (G) GTC Val (V) GCC Ala (A) GAC Asp (D) GGC Gly (G) GTA Val (V)GCA Ala (A) GAA Glu (E) GGA Gly (G) GTG Val (V) GCG Ala (A) GAG Glu (E)GGG Gly (G)

It is to be appreciated that any polynucleotide that encodes apolypeptide in accordance with the invention falls within the scope ofthis invention, regardless of the codons used. The codon usage isadapted for optimized expression in the cells of a given prokaryote oreukaryote.

The polynucleotides encoding Mitrecin A are prepared by incorporatingcodons preferred for use in the genes of a given species into the DNAsequence. Also provided are polynucleotide expression constructs,vectors, host cells comprising nucleic acid fragments of codon-optimizedcoding regions which encode Mitrecin A polypeptides, and various methodsof using the polynucleotide expression constructs, vectors, host cellsto kill or prevent growth of one or more bacteria.

Given the large number of gene sequences available for a wide variety ofanimal, plant and microbial species, it is possible to calculate therelative frequencies of codon usage. Codon usage tables are readilyavailable, for example, at the “Codon Usage Database” available atwww.kazusa.or.jp/codon/ (visited May 30, 2006), and these tables can beadapted in a number of ways. See Nakamura, Y., et al., “Codon usagetabulated from the international DNA sequence databases: status for theyear 2000” Nucl. Acids Res. 28:292 (2000). Codon usage tables forhumans, Escherichia coli, and P. fluorescens calculated from GenBankRelease 151.0, are reproduced below as Tables 10-12 (fromwww.kazusa.or.jp/codon/supra). These tables use mRNA nomenclature, andso instead of thymine (T) which is found in DNA, the tables use uracil(U) which is found in RNA. The tables have been adapted so thatfrequencies are calculated for each amino acid, rather than for all 64codons.

DNA Synthesis

A number of options are available for synthesizing codon optimizedcoding regions designed by any of the methods described above, usingstandard and routine molecular biological manipulations well known tothose of ordinary skill in the art. In one approach, a series ofcomplementary oligonucleotide pairs of 80-90 nucleotides each in lengthand spanning the length of the desired sequence are synthesized bystandard methods. These oligonucleotide pairs are synthesized such thatupon annealing, they form double stranded fragments of 80-90 base pairs,containing cohesive ends, e.g., each oligonucleotide in the pair issynthesized to extend 3, 4, 5, 6, 7, 8, 9, 10, or more bases beyond theregion that is complementary to the other oligonucleotide in the pair.The single-stranded ends of each pair of oligonucleotides are designedto anneal with the single-stranded end of another pair ofoligonucleotides. The oligonucleotide pairs are allowed to anneal, andapproximately five to six of these double-stranded fragments are thenallowed to anneal together via the cohesive single stranded ends, andthen they ligated together and cloned into a standard bacterial cloningvector, for example, a TOPO vector available from InvitrogenCorporation, Carlsbad, Calif. The construct is then sequenced bystandard methods. Several of these constructs consisting of 5 to 6fragments of 80 to 90 base pair fragments ligated together, i.e.,fragments of about 500 base pairs, are prepared, such that the entiredesired sequence is represented in a series of plasmid constructs. Theinserts of these plasmids are then cut with appropriate restrictionenzymes and ligated together to form the final construct. The finalconstruct is then cloned into a standard bacterial cloning vector, andsequenced. Additional methods would be immediately apparent to theskilled artisan. In addition, gene synthesis is readily availablecommercially.

DNA Hybridization

“Hybridization” refers to the association of two nucleic acid sequencesto one another by hydrogen bonding. Typically, one sequence will befixed to a solid support and the other will be free in solution. Then,the two sequences will be placed in contact with one another underconditions that favor hydrogen bonding. Factors that affect this bondinginclude: the type and volume of solvent; reaction temperature; time ofhybridization; agitation; agents to block the non-specific attachment ofthe liquid phase sequence to the solid support (Denhardt's reagent orBlotto); concentration of the sequences; use of compounds to increasethe rate of association of sequences (dextran sulfate or polyethyleneglycol); and the stringency of the washing conditions followinghybridization.

“Stringency” refers to hybridization conditions that favor associationof very similar sequences over sequences that differ. Hybridizationconditions of moderate stringency are as follows: Filters containing DNAare pre-treated for 6 hours at 40° C. in a solution containing 10% to35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP,0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA.Hybridizations are carried out in a solution containing 6×SSC, 50 mMTris-HCl (pH 7.5), 1 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500ug/ml denatured salmon sperm DNA.

Hybridization conditions of high stringency are as follows: Filterscontaining DNA are pre-treated for 6 hours at 40° C. in a solutioncontaining 36% to 50% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mMEDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmonsperm DNA. Hybridizations are carried out in a solution containing6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1%BSA, and 500 ug/ml denatured salmon sperm DNA.

Hybridization conditions of very high stringency are as follows: Filterscontaining DNA are pre-treated for 6 hours at 40° C. in a solutioncontaining 51% to 70% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mMEDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmonsperm DNA. Hybridizations are carried out in a solution containing6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1%BSA, and 500 μg/ml denatured salmon sperm DNA.

Hybridization conditions are controlled such that the probes hybridizeto nucleic acids that are identical to SEQ ID NO: 1. Under certainconditions, the probes also hybridize to nucleic acids that are 95% or99% identical to SEQ ID NO: 1. Alternatively, conditions are such thatthe probes hybridize to nucleic acids at least 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. Typically,suitable probes are fragments which are significantly shorter than thefull length sequence shown in SEQ ID NO: 1. Suitable fragments cancontain from 5 to 100 nucleotides, preferably about 10 to about 80nucleotides. The nucleotide sequence of such fragments is typically atleast 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to aportion of the sequence shown in SEQ ID NO: 1 or its complement.Suitable fragments can contain modified bases such as inosine,methyl-5-deoxycytidine, deoxyuridine, demithylamino-5-deoxyuridine, ordiamino-2,6 purine. Clones in libraries which contain insert genomic DNAfragments encoding a Mitrecin A polypeptide will hybridize to one ormore of the fragments.

A nucleotide sequence encoding a polypeptide at least 90%, 95%, 96%,97%, 98%, 99%, or 100% identical to SEQ ID NO: 2, fragments, analogs,derivatives, or variants described herein is useful for its ability tohybridize selectively, i.e., form duplex molecules with complementarystretches of other polypeptide genes. Depending on the application, avariety of hybridization conditions may be employed to achieve varyingsequence identities. In specific aspects, nucleic acids are providedwhich comprise a sequence complementary to at least 10, 15, 25, 50, 100,200, 250, 300, 350, 400, or 450 nucleotides of a Mitrecin A polypeptidedescribed above. In specific embodiments, nucleic acids which hybridizeto a Mitrecin A protein nucleic acid (e.g. having sequence SEQ ID NO: 1)under annealing conditions of low, moderate or high stringencyconditions are within the scope of the invention.

For a high degree of selectivity, relatively stringent conditions areused to form the duplexes, such as, by way of example and notlimitation, low salt and/or high temperature conditions, such asprovided by hybridization in a solution of salt, e.g., 0.02 M to 0.15 MNaCl at temperatures of between about 50° C. to 70° C. For someapplications, less stringent hybridization conditions are required, byway of example and not limitation such as provided by hybridization in asolution of 0.15 M to 0.9 M salt, e.g., NaCl, at temperatures rangingfrom between about 20° C. to 55° C. Hybridization conditions can also berendered more stringent by the addition of increasing amounts offormamide, to destabilize the hybrid duplex. Thus, particularhybridization conditions can be readily manipulated. By way of exampleand not limitation, in general, convenient hybridization temperatures inthe presence of 50% formamide are: 42° C. for a probe which is 95 to100% homologous to the target fragment, 37° C. for 90 to 95% homologyand 32° C. for 70 to 90% homology. One aspect of the invention isdirected to an isolated polynucleotide comprising a nucleic acidfragment which hybridizes, upon incubation in a solution comprising 50%formamide at about 37° C., to a DNA sequence which is complementary toSEQ ID NO:1 or fragments thereof, or a polynucleotide that is acodon-optimized coding region encoding a polypeptide of SEQ ID NO:2, orfragments thereof, wherein the nucleic acid fragment encodes a MitrecinA polypeptide which is soluble when expressed in E. coli.

Other low, moderate and high stringency conditions are well known tothose of skill in the art, and will vary predictably depending on thebase composition and length of the particular nucleic acid sequence andon the specific organism from which the nucleic acid sequence isderived. For guidance regarding such conditions see, for example,Sambrook et al., Molecular Cloning, A Laboratory Manual, Second Edition,Cold Spring Harbor Press, N.Y., pp. 9.47-9.57 (1989); and Ausubel etal., Current Protocols in Molecular Biology, Green Publishing Associatesand Wiley Interscience, N.Y. (1989).

Vectors, Expression Systems, and Methods of Producing

The present invention further provides a vector comprising apolynucleotide of the present invention. The term “vector,” as usedherein, refers to any of a number of nucleic acids into which a desiredsequence may be inserted by restriction and ligation for transportbetween different genetic environments or for expression in a host cell.Vectors may be DNA or RNA. Vectors include, but are not limited to,plasmids, phage, phagemids, bacterial genomes, and virus genomes andvirus-like particles. A cloning vector is one which is able to replicatein a host cell, and which is further characterized by one or moreendonuclease restriction sites at which the vector may be cut in adeterminable fashion and into which a desired DNA sequence may beligated such that the new recombinant vector retains its ability toreplicate in the host cell. In the case of plasmids, replication of thedesired sequence may occur many times as the plasmid increases in copynumber within the host bacterium or just a single time per host beforethe host reproduces by mitosis. In the case of phage, replication mayoccur actively during a lytic phase or passively during a lysogenicphase. Certain vectors are capable of autonomous replication in a hostcell into which they are introduced. Other vectors are integrated intothe genome of a host cell upon introduction into the host cell, andthereby are replicated along with the host genome.

Any of a wide variety of suitable cloning vectors are known in the artand commercially available which can be used with appropriate hosts. Thevector can be, for example, in the form of a plasmid, a viral particle,a phage, cosmid, etc. As used herein, the term “plasmid” refers to acircular, double-stranded construct made up of genetic material (i.e.,nucleic acids), wherein the genetic material is extrachromosomal andreplicates autonomously. A polynucleotide of the present invention maybe in a circular or linearized plasmid or vector, or other linear DNAwhich may also be non-infectious and nonintegrating (i.e., does notintegrate into the genome of host cells). Procedures for inserting anucleotide sequence into an expression vector, and transforming ortransfecting into an appropriate host cell and cultivating underconditions suitable for expression are generally known in the art, asdescribed generally in Sambrook et al., Molecular Cloning, a LaboratoryManual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989).

Also provided is a vector comprising a nucleic acid sequence encodingfull-length or mature Mitrecin A polypeptides or fragments, variants,derivatives, or analogs thereof described herein. In particular, thepresent invention is directed to a vector comprising a nucleic acidfragment, which encodes a Mitrecin A polypeptide fragment describedherein.

The present invention also includes a vector comprising a polynucleotidewhich encodes a polypeptide comprising, consisting essentially of, orconsisting of an amino acid sequence at least 90%, 95%, 96%, 97%, 98%,99%, or 100% identical to a SEQ ID NO: 2, a fragment thereof, aderivative thereof, or a variant thereof.

In another embodiment, the present invention is directed to a vectorcomprising a polynucleotide comprising, consisting essentially of, orconsisting of a nucleotide sequence encoding a Mitrecin A polypeptidevariant that lacks immunogenicity, wherein said polypeptide retains theantimicrobial activity.

In other embodiments, the present invention is further directed to avector comprising a nucleic acid fragment, where the nucleic acidfragment is a fragment of a codon-optimized coding region operablyencoding any Mitrecin A polypeptides described herein. Furthermore, thepresent invention includes a vector comprising any polynucleotides ofthe present invention.

Additional Streptomyces-derived coding or non-coding regions orendolysin coding regions may also be included on the vector, e.g., aplasmid, or on a separate vector, and expressed, either using nativeMitrecin A codons or codons optimized for expression in the host inwhich the polypeptide is being expressed. When such a vector isdelivered to a host, e.g., to a bacterial, plant or eukaryotic cell, oralternatively, in vivo to a tissue of the animal to be treated orimmunized, the transcriptional unit will thus express the encoded geneproduct. The level of expression of the gene product will depend to asignificant extent on the strength of the associated promoter and thepresence and activation of an associated enhancer element, as well asthe optimization of the coding region.

A variety of host-expression vector systems may be utilized to expressthe polypeptides of the present invention. The term “expression vector”refers to a vector that is capable of expressing the polypeptide of thepresent invention, i.e., the vector sequence contains the regulatorysequences required for polypeptide expression such as promoters,ribosome binding sites, etc. The term “expression” refers to thebiological production of a product encoded by a coding sequence. In mostcases a DNA sequence, including the coding sequence, is transcribed toform a messenger-RNA (mRNA). The messenger-RNA is then translated toform a polypeptide product which has a relevant biological activity.Also, the process of expression may involve further processing steps tothe RNA product of transcription, such as splicing to remove introns,and/or post-translational processing of a polypeptide product.

Vector-host systems include, but are not limited to, systems such asbacterial, mammalian, yeast, insect or plant cell systems, either invivo, e.g., in an animal or in vitro, e.g., in mammalian cell cultures.The selection of an appropriate host is deemed to be within the scope ofthose skilled in the art from the teachings herein.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention as described above.Thus, one aspect of the invention is directed to a host cell comprisinga vector which contains a polynucleotide of the present invention. Theengineered host cell can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the polynucleotides. The culture conditions,such as temperature, pH and the like, are those previously used with thehost cell selected for expression, and will be apparent to theordinarily skilled artisan. The term “transfect,” as used herein, refersto any procedure whereby eukaryotic cells are induced to accept andincorporate into their genome isolated DNA, including but not limited toDNA in the form of a plasmid. The term “transform,” as used herein,refers to any procedure whereby bacterial cells are induced to acceptand incorporate into their genome isolated DNA, including but notlimited to DNA in the form of a plasmid.

A large number of suitable vectors are known to those of skill in theart, and are commercially available. The following bacterial vectors areprovided by way of example: pET, pET28, pBAD, pTrcHIS, pBR322, pQE70,pQE60, pQE-9 (Qiagen), phagescript, psiX174, pBluescript SK, pbsks,pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3,pDR540, pBR322, pPS10, RSF1010, pRIT5 (Pharmacia); pCR (Invitrogen);pLex (Invitrogen), and pUC plasmid derivatives. However, any otherplasmid or vector can be used as long as it is replicable and viable inthe host. In addition, the lambda promoter provides high-levelexpression of recombinant proteins.

A suitable expression vector contains regulatory sequences which can beoperably joined to an inserted nucleotide sequence encoding a Mitrecin Apolypeptide. As used herein, the term “regulatory sequences” meansnucleotide sequences which are necessary for or conducive to thetranscription of an inserted sequence coding a Mitrecin A polypeptide bya host cell and/or which are necessary for or conducive to thetranslation by a host cell of the resulting transcript into the desiredMitrecin A polypeptide. Regulatory sequences include, but are notlimited to, 5′ sequences such as operators, promoters and ribosomebinding sequences, and 3′ sequences such as polyadenylation signals.Regulatory sequences may also include enhancer sequences or upstreamactivator sequences.

Generally, bacterial vectors will include origins of replication andselectable markers, e.g., the ampicillin, tetracycline, kanamycin,resistance genes of E. coli, permitting transformation of the host celland a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promotersinclude, but are not limited to, the T7 promoter, lambda (λ) promoter,T5 promoter, and lac promoter, or promoters derived from operonsencoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK),acid phosphatase, or heat shock proteins, among others.

Once an expression vector is selected, the polynucleotide of theinvention is cloned downstream of the promoter, often in a polylinkerregion. This plasmid is transformed into an appropriate bacterialstrain, and DNA is prepared using standard techniques. The orientationand DNA sequence of the polynucleotide as well as all other elementsincluded in the vector are confirmed using restriction mapping, DNAsequence analysis, and/or PCR analysis. Bacterial cells harboring thecorrect plasmid can be stored as cell banks.

Examples of mammalian host-expression systems include cell lines capableof expressing a compatible vector, for example, the COS, C127, 3T3, CHO,HeLa and BHK cell lines. Examples of suitable expression vectors includepWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL(Pharmacia), p75.6 (Valentis), pCEP (Invitrogen), pCEI (Epimmune),pZERO, pTrc99A, pUC19, pUC18, pKK223-3, pEX1, pCAL, pET, pSPUTK,pTrxFus, pFastBac, pThioHis, pTrcHis, and pLEx, pET-17b, pET-11a,pET-24a-d(+) and pET-9a pK233 (or any of the tac family of plasmids),pT7, lambda pSKF, and pET-28(a)+, vectors useful in yeast cells,including YIp, YRp, YCP, YEp and YLp plasmids as well as viral genomesfrom which to construct viral vectors such as Simian virus 40 (SV40),bovine papilloma virus, pox virus such as vaccinia virus, e.g., VV MVA,and parvovirus, including adeno-associated virus, retrovirus,herpesvirus, adenovirus, retroviral, e.g., murine leukemia virus andlentiviruses (e.g., human immunodeficiency virus), alphavirus, andpicornavirus. References citing methods for the in vivo introduction ofnon-infectious virus genomes to animal tissues are well known to thoseof ordinary skill in the art. Any of a variety of methods known in theart can be used to insert a nucleotide sequence coding for a Mitrecin Apolypeptide into a suitable expression vector.

Generally, mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. Such promoters may also be derived from viralsources, such as, e.g., human cytomegalovirus (CMV-IE promoter), herpessimplex virus type-1 (HSV TK promoter), the adenovirus late promoter;and the vaccinia virus 7.5K promoter, or can be derived from the genomeof mammalian cells (e.g., metallothionein promoter). Nucleic acidsequences derived from the SV40 splice and polyadenylation sites can beused to provide the required nontranscribed genetic elements. A varietyof transcription control regions are known to those skilled in the art.These include, without limitation, transcription control regions whichfunction in animal cells, such as, but not limited to, promoter andenhancer segments from cytomegaloviruses (the immediate early promoter,in conjunction with intron-A), simian virus 40 (the early promoter), andretroviruses (such as Rous sarcoma virus). Other transcription controlregions include those derived from animal genes such as actin, heatshock protein, bovine growth hormone and rabbit β-globin, as well asother sequences capable of controlling gene expression in eukaryoticcells. Additional suitable transcription control regions includetissue-specific promoters and enhancers as well as lymphokine-induciblepromoters (e.g., promoters inducible by interferons or interleukins).Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited toribosome binding sites, translation initiation and termination codons,elements from picornaviruses (particularly an internal ribosome entrysite, or IRES, also referred to as a CITE sequence).

Yeast host-expression systems include a yeast host (e.g., Saccharomyces,Pichia, Hansenula, Kluyveromyces, Schizosaccharomyces, Schwanniomycesand Yarrowia) transformed with recombinant yeast expression vectorscontaining polypeptide coding sequences, employing suitable vectors andcontrol sequences. Suitable yeast expression vectors are known to thosein the art and include, but are not limited to, e.g., pAL19, paR3, pBG1,pDBlet, pDB248X, pEA500, pFL20, pIRT2, pJK148, pON163, pSP1, pSP3,pUR19, pART1, pCHY21, REP41, pYZ1N, pSLF104, pSLF172, pDS472, pSGP572,pSLF1072, REP41MH-N, pFA6a-kanMX6, pARTCM, and pALL.

Insect host systems (e.g., Trichoplusia, Lepidoptera, Spodoptera,Drosophila and Sf9) infected with recombinant expression vectors (e.g.,baculovirus, pDEST™10 Vector (Invitrogen), pMT-DEST48 Vector(Invitrogen), pFastBac Dual (Invitrogen), pIE1-neo DNA (Novagen),pIEX™-1 DNA (Novagen), containing polypeptide coding sequences of thepresent invention are also within the scope of the invention. See e.g.,O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual.Oxford Univ Press (1994).

Plant cell systems (e.g., Arabidopsis) infected with recombinant virusexpression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaicvirus, TMV) or transformed with recombinant plasmid expression vectors(e.g., Ti plasmid) containing polypeptide coding sequences of thepresent invention, containing polypeptide coding sequences are alsowithin the scope of the invention. A list of vectors for a wide varietyof plants can be found athttp://www.arabidopsis.org/servlets/Order?state=catalog (viewed Jun. 20,2006).

One of skill in the art will recognize that some of the above listedvectors are capable of replicating and expressing polypeptides in morethan one type of host, e.g., the pOG44 plasmid can replicate and expresspolypeptides in both prokaryotic and eukaryotic cells.

In one embodiment, production of the polypeptides of the presentinvention can be achieved by culturing the host cells, expressing thepolynucleotides of the present invention, and recovering thepolypeptides. Determining conditions for culturing the host cells andexpressing the polynucleotide are generally specific to the host celland the expression system and are within the knowledge of one of skillin the art. In another embodiment, production of the polypeptides of theinvention includes a synthetic method of making a polypeptide. Thesynthetic methods of making polypeptides are known in the art. Inaddition, appropriate methods for recovering the polypeptide of interestare known to those in the art, and include, but are not limited to,chromatography, filtration, precipitation, or centrifugation.

Compositions

Compositions that contain one or more polypeptides or polynucleotides ofthe invention are a further embodiment of the invention. Compositions ofthe invention also include a vector or a host cell comprising thepolynucleotide of the invention.

The present invention relates to the use of Mitrecin A polypeptidesprovided by the present invention for the reduction of certain bacterialpopulations, including methods and compositions to prevent, inhibit, orreduce bacterial contamination or to treat, prevent, or amelioratevarious bacterial infections. Thus, the present invention also relatesto compositions and formulations comprising Mitrecin A polypeptidesaccording to the present invention and the use of such compositions inprophylaxis or therapy of bacterial diseases, bacterial infections,bacterial contamination, or bacterial colonisations. In one aspect, acomposition or formulation of the present invention is a decontaminationcomposition or decontamination formulation. In another aspect, acomposition or formulation of the present invention is a decolonisationcomposition or decolonisation formulation.

In some aspects, a composition or formulation of the present inventioncan be prepared as cream, lotion, liquid, solid, spray, solution, gel,emulsion, suspension, microemulsion, microcapsule, microgranule, ionicand/or non-ionic follicular dispersion, ointment, stick, or power. Inother aspects, the composition or formulation of the invention can beformulated in the form of injections, oils, moisturizers, aerosols, ornasal inhalers by any method known in the art. These preparations aredescribed in the following formulary known to all pharmaceuticalchemists: Remington's Pharmaceutical Science, 15th Edition, 1975, MackPublishing Company, Easton, Pa. 18042, Chapter 87: Blaug, Seymour.

In accordance with one embodiment of the invention, it may be desirableto contact a surface of a food with a polypeptide of the invention afterprocessing or cooking. Additional embodiments include contacting thepolypeptide of the invention with a non-food surface that is expected tocome into contact with food, for example, a corner, table, ledge,food-processing machinery, or food packaging. This embodiment of theinvention is a composition or formulation comprising a Mitrecin Apolypeptide, a polynucleotide encoding a Mitrecin A polypeptide, avector comprising the polynucleotide, or the host cell comprising thepolynucleotide as a food preservative. Compositions of the inventionwhen used on food or food packaging, for example, can be useful forpreventing or inhibiting spoilage by bacteria on food or for enhancingor extending food storage life compared to a composition which does notcomprise the Mitrecin A polypeptide, the Mitrecin A polynucleotide, thevector, or the host cell. In some embodiments, the composition of thepresent invention can further comprise one or more preservatives, whichare useful for preventing or inhibiting spoilage by bacteria on food orfor enhancing or extending food storage life.

Another embodiment of the invention provides a disinfectant or asanitizing agent comprising a Mitrecin A polypeptide, a polynucleotideencoding a Mitrecin A polypeptide, a vector comprising thepolynucleotide, or a host cell comprising the polynucleotide. Inparticular, a Mitrecin A polypeptide can be used to disinfect orsanitize medical tools, e.g., surgical tools, before or after a medicaltreatment, e.g., surgery, e.g., during hemodialysis. Similarly,premature infants and immune-compromised persons, or those subjects withneed for a medical treatment, e.g., prosthetic devices, can be treatedwith a Mitrecin A polypeptide or polynucleotide of the presentinvention, either prophylactically or during acute infection. In thesame context, nosocomial infections may be treated prophylactically orduring acute phase with a Mitrecin A polypeptide of the presentinvention. For example, a disinfectant or sanitizing agent of theinvention is used to treat or apply the area in which bacteria aregrowing or bacteria are expected to grow, e.g., hospital surfaces,tables, ledges, desks, floors, hospital machinery, and etc. In thisembodiment, a Mitrecin A polypeptide of the present invention may beused as a disinfectant also in combination with other ingredients usefulin a disinfecting solution like detergents, tensids, solvents,antibiotics, lantibiotics, or bacteriocins.

In certain embodiments, a composition of the invention includes anantibiotic comprising a Mitrecin A polypeptide, a polynucleotideencoding the Mitrecin A polypeptide, a vector comprising thepolynucleotide, or a host cell comprising the polynucleotide. In oneaspect, a composition of the invention is used to a subject who issuffering from an infection by pathogenic bacteria or is suspected ofcarrying pathogenic bacteria. In another aspect, a composition of theinvention is used to a subject who is expected to come into a contactwith pathogenic bacteria as a prophylactic measure.

A composition as disclosed herein may comprise more than one Mitrecin Apolypeptide according to the present invention and/or may furthercomprise one or more additional agents. The one or more additionalagents can potentiate the bactericidal activity of a Mitrecin Apolypeptide of the invention or the bacteriostatic activity of aMitrecin A polypeptide. The additional agent can be any agents in anamount that is effective to enhance the antimicrobial effect of aMitrecin A polypeptide of the invention. Non-limiting examples of anadditional agent include an enzyme, an antibiotic, an antifungal agent,an anti-viral agent, a bactericide, an analgesic, a destructive therapyagent, a bacteriocin, and an anti-inflammatory agent.

In some embodiments, a composition or formulation according to thepresent invention comprises a carrier suitable for delivering a MitrecinA polypeptide or polynucleotide to the site of the bacterial growth,bacterial disease, bacterial infection or bacterial colonisation.“Carriers” as used herein include one or more acceptable carriers,excipients or stabilizers, which are non-toxic to the cell or thesubject who may be exposed to a composition of the invention. In oneembodiment, a carrier useful for the invention is a physiologically orpharmaceutically acceptable carrier. In one example, a physiologicallyacceptable carrier is an aqueous pH buffered solution. Examples ofphysiologically acceptable carriers include buffers such as phosphate,citrate, and other organic acids; antioxidants including ascorbic acid;low molecular weight (less than about 10 residues) polypeptide;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as polyoxyethylene sorbitol esters (TWEEN®),polyethylene glycol (PEG), and ethylene oxide and propylene oxide blockcopolymers (PLURONICs®).

In other embodiments, the carrier for a composition of the invention maybe a stabilizing buffer for maintaining a suitable pH range.Compositions of the invention are prepared for storage by mixing theactive ingredient having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions. Any of thecarriers for Mitrecin A polypeptides of the present invention may bemanufactured by conventional means.

The compositions and formulations comprising a Mitrecin A polypeptide ofthe present invention as an active ingredient are applied in aneffective amount when used in prophylaxis and therapy. The term“effective amount” refers to an amount of an active ingredientsufficient to achieve a desired effect without causing an undesirableside effect. In one example, an “effective amount” is an amountsufficient to kill one or more bacteria. In another example, an“effective amount” is an amount sufficient to prevent growth of one ormore bacteria. In some cases, it may be necessary to achieve a balancebetween obtaining a desired effect and limiting the severity of anundesired effect. The amount of active ingredient used will varydepending upon the type of active ingredient and the intended use of thecomposition and/or formulation of the present invention. In a specificexample, an effective amount of a Mitrecin A polypeptide is about 0.1 ngto about 100 mg, about 1 ng to about 50 mg, about 100 ng to about 10 mg,about 1 μg to about 10 mg, about 10 μg to about 1 mg, or about 100 μg toabout 1 mg. In other embodiments, an effective amount of a Mitrecin Apolypeptide is about 15 ng/ml to about 1.5 mg/ml.

In some embodiments, a mild surfactant in an amount effective topotentiate the antimicrobial effect of a Mitrecin A polypeptide may beused in or in combination with a therapeutic or prophylacticcomposition. Suitable mild surfactants include, inter alia, esters ofpolyoxyethylene sorbitan and fatty acids (Tween series), octylphenoxypolyethoxy ethanol (Triton X series), n-Octyl-p-D-glucopyranoside,n-Octyl-β-D-thioglucopyranoside, n-Decyl-D-glucopyranoside,n-Dodecyl-p-D-glucopyranoside, and biologically occurring surfactants,e.g., fatty acids, glycerides, monoglycerides, deoxycholate and estersof deoxycholate.

A mode of delivery of the composition comprising a Mitrecin Apolypeptide includes, but is not limited to a smear, spray, a drink, apill, a gargle, a chewing gum, a dietary supplement, a time-releasepatch, a liquid absorbed wipe, and combinations thereof.

One mode of delivery of the composition comprising a Mitrecin Apolypeptide is a smear. Another mode of delivery of the compositioncomprising a Mitrecin A polypeptide is a spray. In some embodiments, amode of delivery of the composition comprising a Mitrecin A polypeptideis a liquid absorbed wipe.

In some embodiments, preservatives may also be used in this inventionand may comprise, for example, about 0.05% to 0.5% by weight of thetotal composition. The use of preservatives assures that if the productis microbially contaminated, the formulation will prevent or diminishmicroorganism growth. Some preservatives useful in this inventioninclude methylparaben, propylparaben, butylparaben, chloroxylenol,sodium benzoate, DMDM Hydantoin, 3-lodo-2-Propylbutyl carbamate,potassium sorbate, chlorhexidine digluconate, or a combination thereof.

Compositions of the present invention can be formulated according toknown methods. Suitable preparation methods are described, for example,in Remington's Pharmaceutical Sciences, 16th Edition, A. Osol, ed., MackPublishing Co., Easton, Pa. (1980), and Remington's PharmaceuticalSciences, 19th Edition, A. R. Gennaro, ed., Mack Publishing Co., Easton,Pa. (1995). Although the composition may be administered as an aqueoussolution, it can also be formulated as an emulsion, gel, solution,suspension, lyophilized form, or any other form known in the art. Inaddition, the composition may contain pharmaceutically acceptableadditives including, for example, diluents, binders, stabilizers, andpreservatives. Once formulated, the compositions of the invention can beadministered directly to the subject. The subjects to be treated can beanimals; in particular, human subjects can be treated.

Methods of Using and Regimens

The present invention includes a method of killing or preventing growthof one or more bacteria comprising contacting a Mitrecin A polypeptidewith the one or more bacteria. The one or more bacteria can be presenton a surface of an object, a plant, or an animal or within the object,plant, or animal. For example, the one or more bacteria, e.g.,pathogenic bacteria, can be killed, or their growth can be inhibited, byapplying a Mitrecin A polypeptide, a polynucleotide encoding theMitrecin A polypeptide, a vector comprising the polynucleotide, a hostcell comprising the vector, or a composition comprising the polypeptideor the polynucleotide on the surface of an object, a plant, or ananimal. The object for example can be an environmental object, e.g.,household furniture, e.g., a bathroom sink, or a hospital area, e.g., ahospital counter. In some aspects, the object is an inanimate item, forexample, floors and/or flooring materials (e.g., Linoleum, tile, carpet,wood, paint, etc.), walls and/or wall covering materials (e.g., paint,wall paper, Formica, tile, etc.) windows and/or window materials (glass,Plexiglas, polycarbonate, etc.), water delivery system components (e.g.,pumps, pipes, reservoirs), structural and/or cosmetic building materials(e.g., concrete, masonry, stucco, steel, stainless steel, aluminum,copper, nickel, cast iron, plastic, fiberglass, carbon fiber, Kevlar)counters, furniture, clothes, dishes, or combinations thereof.

In one aspect, the present invention is directed to a method ofpreventing, ameliorating, or treating a disease or disorder in a plantcomprising contacting the plant with an effective amount of a Mitrecin Apolypeptide, a polynucleotide encoding the Mitrecin A polypeptide, avector comprising the polynucleotide, a host cell comprising thepolynucleotide, or a composition comprising the Mitrecin A polypeptide,wherein the polypeptide, the polynucleotide, the vector, the host cell,or the composition prevents, ameliorates, or treats the disease ordisorder in the plant. The disease or disorder in the plant may inducegrowth inhibition or retardation, less fruit formation, or a higherdeath rate of the plant compared to a plant without the disease ordisorder.

In another aspect, the invention includes a method of promoting plantgrowth or disease resistance in a plant comprising contacting a MitrecinA polypeptide, a polynucleotide encoding the Mitrecin A polypeptide, avector comprising the polynucleotide, a host cell comprising thepolynucleotide, or a composition comprising a Mitrecin A polypeptide,wherein the polypeptide, the polynucleotide, the vector, the host cell,or the composition promotes plant growth or disease resistance in theplant.

In other aspects, the method further comprises assessing prevention,amelioration, or treatment of a plant disease or disorder or assessingthe plant growth promoting activity or plant disease resistancepromoting activity.

A composition comprising a Mitrecin A polypeptide or a Mitrecin Apolynucleotide can be formulated to be suitable for application to aplant. In one aspect, the composition is used to promote plant growthand/or promote disease resistance in plants. As used herein, the term“plant growth promoting activity” encompasses a wide range of improvedplant properties, including, without limitation, improved nodulation(e.g. increased number of nodules), nitrogen fixation (e.g. increasednitrogen concentration as measured by mg g⁻¹ dry weight of plantmaterial), increased leaf area, increased seed germination, increasedleaf greenness (e.g. as measured by SPAD), increased photosynthesis(μmol cm⁻² s⁻¹), or an increase in accumulated dry-weight of the plant.

As used herein, the term “plant disease resistance promoting activity”or the like, encompasses, without limitation, increased resistance topathogen attack or increased production of one or more secondarymetabolites that function to improve the resistance of a plant topathogen attack, as discussed herein.

An increase or improvement in plant growth or disease resistance means astatistically significant increase or improvement in the measuredcriterion of plant growth or disease resistance in a plant treated witha polypeptide according to the invention relative to an untreatedcontrol plant.

Assessment of the plant growth promoting activity of polypeptides may beaccomplished by known methods. For instance, a polypeptide of interestmay be applied by leaf spray or root irrigation to test plants, such assoybean plants. Plants may then be grown under controlled environmentconditions (growth chamber or greenhouse) for e.g. about 40 days. Atharvest, data may be collected concerning e.g. plant height, leafgreenness, leaf area, nodule number, nodule dry weight, shoot and dryroot weight or length, nitrogen content and photosynthesis and comparedto controls.

Assessment of plant disease resistance promoting activity ofpolypeptides may also be accomplished by known methods, such as bydetecting or measuring a reduction in pathogen infestation of a plant,or indirectly by detecting or measuring increased production of one ormore secondary metabolites that function to improve the resistance of aplant to pathogen attack. Exemplary secondary metabolites includelignification-related enzymes such as phenylalanine ammonia lyase (PAL),and tyrosine ammonia lyase (TAL), antioxidative enzymes such asperoxidase (POD), catalase (CAT), and superoxide dismutase (SOD), andtotal phenolic compounds. Various methods for detecting or measuringincreases in enzyme activity levels in plants (e.g. PAL, TAL, POD, CADand SOD) are known in the art and exemplary techniques are described inthe examples herein. Similarly, techniques for determiningconcentrations or levels of total phenolic compounds are known andexemplary methods are described in the examples herein.

An increase or improvement in plant growth or disease resistance means astatistically significant increase or improvement in the measuredcriterion of plant growth or disease resistance in a plant treated witha polypeptide according to the invention relative to an untreatedcontrol plant.

In certain aspects, the present invention provides a method ofpreventing, ameliorating, or treating a disease or disorder in an animalcomprising contacting the animal with an effective amount of a MitrecinA polypeptide, a polynucleotide encoding the Mitrecin A polypeptide, avector comprising the polynucleotide, a host cell comprising thepolynucleotide, or a composition comprising the Mitrecin A polypeptideor the polynucleotide, wherein the polypeptide, the polynucleotide, thevector, the host cell, or the composition prevents, ameliorates, ortreats the disease or disorder in the animal.

A Mitrecin A polypeptide or a composition comprising the Mitrecin Apolypeptide according to the present invention is useful for treatingand eliminating bacterial infestations anywhere, including upperrespiratory infections, topical and systemic infections, vaginalinfections, eye infections, ear infections, infections requiringparenteral treatment, as well as for the elimination of bacteria on anysurface, including human skin and mucous membrane, e.g., the mucousmembrane of the upper respiratory tract, e.g., the mucous membrane ofthe nasal cavity. A Mitrecin A polypeptide or a composition comprisingthe Mitrecin A polypeptide according to the present invention areparticularly useful for the prophylaxis and treatment of upperrespiratory infections, skin infections, wounds, burns, vaginalinfections, eye infections, intestinal disorders and dental disorders.Specifically, the invention provides the application of Mitrecin Apolypeptides for nasal and/or skin decolonisation of human and animals.

A Mitrecin A polypeptide of the invention may be administered to anysubject afflicted with, diagnosed as afflicted with, or suspected ofbeing afflicted with, an infection or contamination by bacteriasusceptible to the Mitrecin A polypeptide. The invention also providesfor the treatment or prevention of an opportunistic infection, such asthat resulting from an undesirable growth of bacteria that are presentin the microbial flora of a human subject or a non-human animal. Anopportunistic infection may be the result of an immunosuppressedcondition in a subject or the result of antibiotic treatment that alterthe commensal flora of the genitourinary (GU) or gastrointestinal (GI)tract. Thus the disclosure also provides for the treatment orprophylaxis of immunosuppressed subjects and subjects exposed to otherpharmaceutical agents. A Mitrecin A polypeptide with its anti-bacterialactivity may be used in combination with another anti-bacterial oranti-microbial agent, such as an antibiotic or anti-fungal agent asnon-limiting examples. An “anti-microbial agent” is an agent or compoundthat can be used to inhibit the growth of, or to kill, single-celledorganisms. Anti-microbial agents include antibiotics, chemotherapeuticagents, antibodies (with or without complement), and chemical inhibitorsof DNA, RNA, protein, lipid, or cell wall synthesis or functions.

Additionally, and in anticipation of a possible emergence of bacterialresistance to a Mitrecin A polypeptide, there can be a concomitantcompromise of the organisms' virulence or fitness where the Mitrecin Apolypeptide targets the virulence or fitness factor of the targetedbacteria. Because a major, but non-limiting, mechanism by which abacterium may become resistant to a Mitrecin A polypeptide is the lossof its receptor for the Mitrecin A polypeptide, the targeting of avirulence or fitness factor as disclosed herein provides many advantagesover traditional antibiotics and bacteriophages. The resistance totraditional antibiotics and bacteriophages can result from manydifferent mechanisms other than loss of the receptor or target moleculeof the antibacterial agent. As non-limiting examples, a Mitrecin Apolypeptide of the invention would not be subject to a bacterial effluxpump to remove the Mitrecin A polypeptide from the cellular environmentand would not be subject to a bacterial nucleic acid deactivationmechanism.

In one aspect of the present invention, Mitrecin A polypeptides areapplied in a method for the treatment or prophylaxis of one or morebacteria, which are selected from the group consisting of aGram-positive bacterium, a Gram-negative bacterium, or both. Examples ofGram-positive target bacteria that can be killed or inhibited include,but are not limited to, Streptomyces avermitilis, Streptomycescoelicolor, Streptomyces lividans, Streptomyces venezuelae, Nocardiasalmonicida, Nocardia vaccinii, Rhodococcus marinonascens, Bacillusmegaterium, Bacillus subtilis, Bacillus cereus, Enterococcus faecalis,Micrococcus luteus, Staphylococcus aureus, Streptococcus sp.,Streptococcus pyogenes, Listeria monocytogenes, Clostridium perfringens,Clostridium botulinum, Lactococcus cremoris, Lactobacillus sp., andLeuconostoc sp. Thus, a Gram-positive target bacterium can be selectedfrom the group consisting of Streptomyces avermitilis, Streptomycescoelicolor, Streptomyces lividans, Streptomyces venezuelae, Nocardiasalmonicida, Nocardia vaccinii, Rhodococcus marinonascens, Bacillusmegaterium, Bacillus subtilis, Bacillus cereus, Enterococcus faecalis,Micrococcus luteus, Staphylococcus aureus, Streptococcus sp.,Streptococcus pyogenes, Listeria monocytogenes, Clostridium perfringens,Clostridium botulinum, Lactococcus cremoris, Lactobacillus sp., andLeuconostoc sp.

Examples of Gram-negative target bacteria include, but are not limitedto, Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium,Salmonella enterica, Campylobacter jejuni, Yersinia enterocolitica,Yersinia pseudotuberculosis, Vibrio cholerae, Vibrio parahaemolyticus,Vibrio vulnificus, Aeromonas hydrophila, Plesiomonas shigelloides,Shigella sonnei, Shigella flexneri, Enterobacter aerogenes,Flavobacterium sp., Acinetobacter sp., and Proteus sp. Thus, aGram-negative target bacterium can be selected from the group consistingof Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium,Salmonella enteritidis, Campylobacter jejuni, Yersinia enterocolitica,Yersinia pseudotuberculosis, Vibrio cholerae, Vibrio parahaemolyticus,Vibrio vulnificus, Aeromonas hydrophila, Plesiomonas shigelloides,Shigella sonnei, Shigella flexneri, Enterobacter aerogenes,Flavobacterium sp., Acinetobacter sp., and Proteus sp.

In some embodiments, the target bacterium is Vibrio sp., Salmonella sp.,Yersinia sp., or Bacillus sp. In a particular embodiment, the targetbacterium is Vibrio cholerae, Salmonella enterica, Yersiniapseudotuberculosis, Shigella sonnei, Aeromonas hydrophila or Bacillussubtilis. In other embodiments, the target bacterium is S. aureus, S.epidermidis, S. haemolyticus, S. simulans, S. saprophytics, S.chromogenes, S. hyicus, S. warneri and/or S. xylosus. The subject may bea human subject or an animal, in particular animals used in livestockfarming and/or dairy farming such as cattle and pigs. The method oftreatment encompasses the application of a Mitrecin A polypeptide of thepresent invention to the site of infection or site to beprophylactically treated against infection in a sufficient amount. Inparticular, the method of treatment may be for the treatment orprophylaxis of infections, in particular by Staphylococcus aureus, ofthe skin, of soft tissues, of bacteremia and/or endocarditis.

Other aspects of the present invention includes a method of detecting ormeasuring presence of one or more pathogenic bacteria in a samplecomprising contacting an effective amount of a Mitrecin A polypeptide, apolynucleotide encoding the Mitrecin A polypeptide, a vector comprisingthe polynucleotide, a host cell comprising the polynucleotide, or acomposition comprising the Mitrecin A polypeptide. In one embodiment,the sample is a tissue of an animal or a plant. In another embodiment,the sample is an environmental sample, e.g., soil or water. In oneembodiment, target bacteria can be captured using a Mitrecin Apolypeptide and detected with a secondary reagent such as an antibody.Suitable targets include, but are not limited to, the targets in Table4.

TABLE 4 Examples of Target Pathogen Target Pathogen Disease Bacillusanthracis Anthrax Multi-Drug Resistant Staphylococcus aureus MRSAYersinia pestis Plague Francisella tularensis Tularemia Vibrio choleraeCholera Salmonella enterica Salmonellosis Shigella sonnei ShigellosisAeromonas hydrophila Gastroenteritis

In some embodiments, a Mitrecin A polypeptide of the invention isformulated with a “pharmaceutically acceptable” excipient or carrier.Such a component is one that is suitable for use with humans, animals,and/or plants without undue adverse side effects. Non-limiting examplesof adverse side effects include toxicity, irritation, and/or allergicresponse. The excipient or carrier is typically one that is commensuratewith a reasonable benefit/risk ratio. In many embodiments, the carrieror excipient is suitable for topical or systemic administration.Non-limiting pharmaceutically suitable carriers include sterile aqueousor non-aqueous solutions, suspensions, and emulsions. Examples include,but are not limited to, standard pharmaceutical excipients such as aphosphate buffered saline solution, water, emulsions such as oil/wateremulsion, and various types of wetting agents. Examples of non-aqueoussolvents are propylene glycol, polyethylene glycol, vegetable oils suchas olive oil, and injectable organic esters such as ethyl oleate.Aqueous carriers include water, alcoholic/aqueous solutions, emulsionsor suspensions, including saline and buffered media. Parenteral vehiclesinclude sodium chloride solution, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's or fixed oils. Intravenous vehicles includefluid and nutrient replenishers, electrolyte replenishers (such as thosebased on Ringer's dextrose), and the like.

A Mitrecin A polypeptide of the invention is typically used in an amountor concentration that is “safe and effective,” which refers to aquantity that is sufficient to produce a desired therapeutic responsewithout undue adverse side effects like those described above. AMitrecin A polypeptide may also be used in an amount or concentrationthat is “therapeutically effective,” which refers to an amount effectiveto yield a desired therapeutic response, such as, but not limited to, anamount effective to slow the rate of bacterial cell division, or tocause cessation of bacterial cell division, or to cause death ordecrease rate of population growth of the bacteria. The safe andeffective amount or therapeutically effective amount will vary withvarious factors but may be readily determined by the skilledpractitioner without undue experimentation. Non-limiting examples offactors include the particular condition being treated, the physicalcondition of the subject, the type of subject being treated, theduration of the treatment, the nature of concurrent therapy (if any),and the specific formulations employed.

A Mitrecin A polypeptide of the disclosure may be administered to asubject by any suitable means. Non-limiting examples include topical orlocalized administration as well as pulmonary (inhalation),gastrointestinal, by catheter or drip tube, or systemic administrationto a subject. Representative, and non-limiting, examples of systemicadministration include intraperitoneal and intravenous administration.In some embodiments, contact between a Mitrecin A disclosed herein and atarget bacterial population results in a decrease in the population ofat least 10, at least 100, at least 1000, or at least 10,000, or more,fold decrease relative to the absence of the bacteriocin. In otherembodiments, the contact may result in a decrease in detectability ofthe bacteria by at least 5, at least 10, at least 20, at least 30, atleast 40, or at least 50, or more, fold relative to the absence of thebacteriocin.

In some embodiments, means of application of Mitrecin A polypeptide(s)of the invention include, but are not limited to, direct, indirect,carrier and special means or any combination of means. Directapplication of a Mitrecin A polypeptide may be by nasal sprays, nasaldrops, nasal ointments, nasal washes, nasal injections, nasal packings,bronchial sprays and inhalers, or indirectly through use of throatlozenges, mouthwashes or gargles, or through the use of ointmentsapplied to the nasal nares, or any combination of these and similarmethods of application. The forms in which a Mitrecin A polypeptide maybe administered include but are not limited to powders, sprays, liquids,gels, ointments, and aerosols. It is most probable that exposure to thebacteria will be through the nose. Preferred are sprays, liquids, gels,ointments, and aerosols.

The dosage and route of administration used in a method of treatment (orprophylaxis) according to the present invention depends on the specifictarget bacteria. The route of administration may be, for example, oral,topical, nasopharyngeal, parenteral, intravenous, rectal or any otherroute of administration.

The effective dosage rates or amounts of a Mitrecin A polypeptide(s) totreat the infection will depend in part on whether the Mitrecin Apolypeptide will be used therapeutically or prophylactically, theduration of exposure of the recipient to the infectious bacteria, thesize and weight of the individual, etc. The duration for use of thecomposition containing a Mitrecin A polypeptide also depends on whetherthe use is for prophylactic purposes, wherein the use may be hourly,daily or weekly, for a short time period, or whether the use will be fortherapeutic purposes wherein a more intensive regimen of the use of thecomposition may be needed, such that usage may last for hours, days orweeks, and/or on a daily basis, or at timed intervals during the day.Any dosage form employed should provide for a minimum number of unitsfor a minimum amount of time. The concentration of the active units ofchimeric polypeptide that may provide for an effective amount or dosageof a Mitrecin A polypeptide may be in the range of 10 units/ml to500,000 units/ml of fluid in the wet or damp environment of the nasaland oral passages, and topically as well and possibly in the range of10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 units/ml to 50,000 units/ml.Representative values thus include about 200 units/ml, 300 units/ml, 500units/ml, 1,000 units/ml, 2,500 units/ml, 5,000 units/ml, 10,000units/ml, 20,000 units/ml, 30,000 units/ml, and 40,000 units/ml. Morespecifically, time exposure to the active polypeptide units mayinfluence the desired concentration of active polypeptide units per ml.It should be noted that carriers that are classified as “long” or “slow”release carriers (such as, for example, certain nasal sprays orlozenges) could possess or provide a lower concentration of active(enzyme) units per ml, but over a longer period of time, whereas a“short” or “fast” release carrier (such as, for example, a gargle) couldpossess or provide a high concentration of active (enzyme) units per ml,but over a shorter period of time. The amount of active units per ml andthe duration of time of exposure depend on the nature of infection,whether treatment is to be prophylactic or therapeutic, and othervariables. Thus, the number of dosages will be dependent upon thecircumstances and can range from 1 to 4 times per day or more, withdurations from one day to multiple weeks.

The route of administration is in accordance with known methods. Whentreating a bacterial exposure or infection, a Mitrecin A polypeptide maybe administered in any suitable fashion, including topicaladministration or through the oral or nasal cavity. For topicalapplication a polypeptide of the present invention may be administeredby way of a lotion or plaster. For nasopharyngeal application a MitrecinA polypeptide according to the present invention may be formulated insaline in order to be applied via a spray to the nose. Dosages anddesired drug concentrations of pharmaceutical compositions of thepresent invention may vary depending on the particular use envisioned.The determination of the appropriate dosage or route of administrationis well within the skill of an ordinary physician.

Kits

The polypeptide or polynucleotide compositions of this invention can beprovided in kit form together with a means for administering thepolypeptide, polynucleotide, or composition of the present invention. Insome embodiments, the kit can further comprise instructions for vaccineadministration.

Typically the kit would include desired composition(s) of the inventionin a container, e.g., in unit dosage form and instructions foradministration. Means for administering the composition of the presentinvention can include, for example, a sterile syringe, an aerosolapplicator (e.g., an inhaler or any other means of nasal or pulmonaryadministration), a gel, a cream, a transdermal patch, transmucosal patch(or any other means of buccal or sublingual administration), or an oraltablet. In some embodiments, the kit of the present invention containstwo or more means for administering the polypeptides, polynucleotides,vectors, or compositions of the present inventions, e.g., two or moresyringes.

In some embodiments, the kit may comprise more than one containercomprising the polypeptide, polynucleotide, or composition of thepresent invention. For example, in some embodiments the kit may comprisea container containing a priming component of the present invention, anda separate container comprising the boosting component of the presentinvention.

Optionally associated with such container(s) can be a notice or printedinstructions. For example, such printed instructions can be in a formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of the manufacture, use or sale for humanadministration of the present invention. “Printed instructions” can be,for example, one of a book, booklet, brochure or leaflet.

The kit can also include a storage unit for storing the components(e.g., means of administering, containers comprising the polypeptides,polynucleotides, or compositions of the present inventions, printedinstructions, etc.) of the kit. The storage unit can be, for example, abag, box, envelope or any other container that would be suitable for usein the present invention. Preferably, the storage unit is large enoughto accommodate each component that may be necessary for administeringthe methods of the present invention.

The present invention can also include a method of delivering apolypeptide, polynucleotide, or composition of the present invention toan animal such as a human in need thereof, the method comprising (a)registering in a computer readable medium the identity of anadministrator (e.g., a physician, physician assistant, nursepractitioner, pharmacist, veterinarian) permitted to administer thepolypeptide, polynucleotide, vector, or composition of the presentinvention; (b) providing the human with counseling informationconcerning the risks attendant the polypeptide, polynucleotide, vector,or composition of the present invention; (c) obtaining informed consentfrom the human to receive the polypeptide, polynucleotide, vector, orcomposition of the present invention despite the attendant risks; and(e) permitting the human access to the polypeptide, polynucleotide,vector, or composition of the present invention.

Having now generally described the inventive subject matter, the samewill be more readily understood through reference to the followingexamples which are provided by way of illustration, and are not intendedto be limiting of the disclosure, unless specified.

EXAMPLES Example 1: Bacterial Strains, Media, and Bacteriocin Production

Streptomyces, a medically and industrially important genus of soilbacteria, produce many useful antibiotics and enzymes, contain largeprokaryotic genomes, and produce secondary metabolites with exceptionalfunctionality. The previously uncharacterized Streptomyces sp. strain212 was isolated on environmental extract medium from a soil sample ofRainbow Bluff, a woodland bluff of Lynn, Ala. The strain was one of 45isolates demonstrating lytic activity against heat-killed bacterialsubstrate. One of the lytic enzyme producers, strain 212, resembled thegenus Streptomyces in colony morphology, carbohydrate utilization, and16S rRNA gene sequence. The total bacteriocin activity of strain 212 wasmeasured against both Gram-negative and Gram-positive bacteria usingzymogram (renaturing SDS-PAGE) analysis (FIG. 1) and the line inoculumassay.

Streptomyces, sp. strain 212 was monitored over three weeks for itscarbohydrate utilization. The carbohydrate utilization assay was used todefine the metabolic capabilities of this bacterium as compared to otherStreptomyces species. Table 5 shows various carbohydrate consumptions byStreptomyces sp. strain 212 during three weeks.

TABLE 5 Carbohydrate Consumption Carbohydrates Week 1 Week 2 Week 3Adonitol − − − Arabinose + + + Dextrin +/− + + Dextrose + + + Fructose− + + Galactose + + + Inositol − − + Inulin +/− + + Lactose − − −Maltose + + + Mannitol − + + Mannose + + + Sorbitol − − − Xylose + + +

Example 2: Phylogenetic Analysis of Streptomyces sp. Strain 212 16S rRNASequence

Phylogenetic relatedness of Strain 212 to other closely related bacteriawas assessed using partial 16S rRNA gene sequences identified withBLASTn. The reference sequences and strain 212 sequence were aligned inBioEdit Sequence Alignment Editor using CLUSTAL W. The neighbor-joiningalgorithm of PAUP* version 4.0 was used to infer the phylogeneticrelatedness of the sequences. Tree topologies were calculated bybootstrap analyses based on 1000 resamplings. FIG. 2 shows theneighbor-joining cladogram based on this analysis which relatedStreptomyces sp. strain 212 and other Streptomyces species.

Example 3: Genome Sequencing and Annotation

Genomic DNA from Strain 212 was subjected to de novo genome sequencingand assembly at The Institute for Genome Sciences (IGS) GenomicsResource Center at the University of Maryland using the 454 GS/FLXpyrosequencing platform and GS de novo sequence assembly software,Newbler. The putative genes within the draft-quality genome wereannotated using the IGS Annotation Engine. Open reading frames (ORFs)were identified by Glimmer 3 algorithm, while tRNA and rRNA genes weredetected by tRNAscan-SE and RNAmmer, respectively. The genome of thestreptomycete, estimated by pyrosequencing, is approximately 10 Mbp witha GC content of 68%. Comparison of the Mitrecin A gene sequence to otherknown genes using BLASTn indicates similarity to bacteriophage endolysingenes.

Example 4: Gene Synthesis, Expression, and Mitrecin A Purification

Annotation of the Streptomyces sp. strain 212 genome identified a suiteof putative bacteriolytic genes, including the gene for Mitrecin A. Thegene for Mitrecin A was fused with a C-terminal 6-histidine tagsequence. Mitrecin A was subsequently synthesized and expressed as seenin FIG. 3A. The first step in the isolation was a partial purificationby one-step immobilized metal ion affinity chromatography to a purity of90%, as estimated using the 2100 Bioanalyzer platform. Partiallypurified protein was stored at −80° C. in 50 mM Tris (pH 9.0), 0.5 mML-arginine, and 10% glycerol. Mitrecin A was further purified using anHPLC system fitted with a Superdex 75 size exclusion column. Mitrecin Awas determined to be isolated from contaminates using 12% SDS-PAGE,western blot, and Bioanalyzer analyses. The N-terminus of the purifiedenzyme was sequenced using Edman degradation at the Iowa StateUniversity Protein Facility. FIG. 3B shows the 14.3 kDa purified proteinas visualized by an anti-His western blot.

Example 5: Effects of Temperature, Salinity, and pH on Mitrecin AActivity

For each stressor, residual enzyme activity was assessed using amodified version of the quantitative dye-release assay described by Zhouet al. using Y. pseudotuberculosis as cell substrate (Zhou et. al,Analytical Biochemistry 171:141-144 (1988)). Thermal stability ofMitrecin A was tested after challenge against various temperatures(4-95° C.) for 30 min, and the results are displayed in FIG. 4A. FIGS.4B and 4C show the results of testing the stability of the enzyme invarious pH and saline conditions in dye-release assays over 16 hincubations at 37° C. Mitrecin A displays the greatest bacteriolyticactivity at elevated pH values ranging from 7 to 11, in salineconditions of 1.5%, and at ambient temperatures.

Example 6: Functional Assays of Mitrecin A Activity

The spectrum of bacteriolytic activity against various Gram-positive andGram-negative bacteria was assessed using slide diffusion assays, anexample of which is shown in FIG. 5. The residual viability of the Y.pseudotuberculosis indicator organism in the presence of variousconcentrations of Mitrecin A was also assayed by serial dilution andcolony forming unit assays after 16 h incubation at 37° C., as shown inFIG. 6. Table 6 summarizes the inhibitory spectrum of Mitrecin A, whichincludes bacteriolytic activity against the medically important generaof Salmonella, Vibrio, and Yersinia.

TABLE 6 Inhibitory Spectrum of Mitrecin A Gram-Positive Gram-NegativeBacteria Lysis Bacteria Lysis Francisella philomiragia Negative Bacilluscereus Negative Salmonella enterica Positive Bacillus thuringiensisNegative Vibrio cholerae Positive Bacillus subtilis Negative YersiniaPositive Staphylococcus aureus Negative pseudotuberculosis Aeromonashydrophila Positive Shigella sonnei Positive Escherichia coli DH5αPositive

Although the present invention has been described in detail withreference to examples above, it is understood that various modificationscan be made without departing from the spirit of the invention.Accordingly, the invention is limited only by the following claims. Allcited patents, patent applications and publications referred to in thisapplication are herein incorporated by reference in their entirety.

What is claimed is:
 1. A method of ameliorating or treating a disease ordisorder in an animal, the method comprising contacting the animal witha formulation comprising an effective amount of an isolated polypeptidecomprising an amino acid sequence at least 90% identical to thefull-length sequence of SEQ ID NO: 2 or a fragment thereof thatcomprises amino acids 54 to 73 (Conserved Region 1) of SEQ ID NO: 2,wherein contacting the animal with the formulation ameliorates or treatsthe disease or disorder in the animal, and wherein the disease ordisorder is an infection caused by a Gram negative bacteria.
 2. Themethod of claim 1, wherein the isolated polypeptide or fragment isconjugated to a second polypeptide.
 3. The method of claim 1, whereinthe amino acid sequence of the isolated polypeptide is that of SEQ IDNO:
 2. 4. The method of claim 1, wherein the amino acid sequence of theisolated polypeptide is that of the fragment of SEQ ID NO: 2 thatcomprises amino acids 54 to 73 (Conserved Region 1) of SEQ ID NO:
 2. 5.The method of claim 4, wherein the fragment further comprises aminoacids 81 to 90 (Conserved Region 2) of SEQ ID NO: 2, amino acids 106 to121 (Conserved Region 3) of SEQ ID NO: 2, or both.
 6. The method ofclaim 1, wherein the Gram-negative bacteria comprise at least one memberof the group consisting of Escherichia coli, Klebsiella pneumoniae,Salmonella typhimurium, Salmonella enterica, Campylobacter jejuni,Yersinia enterocolitica, Yersinia pseudotuberculosis, Vibrio cholerae,Vibrio parahaemolyticus, Vibrio vulnificus, Aeromonas hydrophila,Plesiomonas shigelloides, Shigella sonnei, Shigella flexneri,Enterobacter aerogenes, Flavobacterium sp., Acinetobacter sp., Shigellasp., Aeromonas sp., and Proteus sp.
 7. The method of claim 1, whereinthe infection is an upper respiratory infection, skin infection, woundinfection, burn infection, vaginal infection, eye infection, intestinaldisorder, or dental disorder.
 8. The method of claim 1, wherein theanimal is a human.
 9. The method of claim 1, wherein the animal is anon-human animal.
 10. The method of claim 1, wherein the formulation issterile.
 11. The method of claim 1, wherein the formulation isformulated as a topical cream or lotion.
 12. The method of claim 1,wherein the formulation is formulated as a gargle, a pill, or a chewinggum.
 13. The method of claim 1, wherein the formulation is formulated asa drink or a dietary supplement.
 14. The method of claim 6, wherein theGram-negative bacteria comprise at least one member of the groupconsisting of Salmonella enterica, Yersinia pseudotuberculosis, Shigellasonnei, and Aeromonas hydrophila.
 15. The method of claim 1, wherein theformulation is formulated as a spray, a powder, or a wipe.